Method for producing aldehyde from co2

ABSTRACT

The invention provides recombinant microorganisms capable of producing isobutyraldehyde using CO 2  as a carbon source. The invention further provides methods of preparing and using such microorganisms to produce isobutyraldehyde.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT/US2009/068863, filed Dec. 18,2009, which claims priority to U.S. Provisional Application No.61/139,593, filed Dec. 20, 2008, and U.S. Provisional Application No.61/219,322, filed Jun. 22, 2009, the disclosures of all of which areincorporated herein by reference in their entirety.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided intext format in lieu of a paper copy and is hereby incorporated byreference into the specification. The name of the text file containingthe sequence listing is 37267_SEQ.txt. The text file is 289 KB; wascreated on Jun. 20, 2011; and is being submitted via EFS-Web with thefiling of the specification.

BACKGROUND

According to the US Energy Information Administration (EIA, 2007), worldenergy-related CO₂ emissions in 2004 were 26,922 million metric tons andincreased 26.7% from 1990. As a result, atmospheric levels of CO₂ haveincreased by about 25% over the past 150 years. Thus, it has becomeincreasingly important to develop new technologies to reduce CO₂emissions.

The world is also facing costly gas and oil and limited reserves ofthese precious resources. Biofuels have been recognized as analternative energy source. While efforts have been made to improvevarious production, further developments are needed.

SUMMARY

The disclosure describes the construction of a novel metabolic systemfor conversion of CO₂ to various higher alcohols using photosyntheticmicroorganism such as, for example, cyanobacteria (Synechococcuselongatus PCC7942). There is currently no known method for production ofhigher alcohols from CO₂.

The disclosure describes the production of biofuels using metabolicallyengineered organisms that can utilize CO₂ as a starting material. Anexemplary pathway is shown in FIG. 1A. This system can be used in anynumber of photosynthetic microorganisms. For example, one such organismis obtained from cyanobacteria. Large scale photobiorection can beperformed on such bacteria. Using techniques described herein,isobutanol production from flask-fermentation using engineered strainsof the disclosure were achieved.

The disclosure provides a recombinant photosynthetic organism comprisinga pathway that converts CO₂ to metabolic intermediates that can be usedin biofuel production. In one embodiment, a pathway starts from CO₂,which produces carbohydrate and various 2-keto acids via the CalvinCycle, photosynthesis and amino acid pathways. The keto acid isconverted to corresponding alcohols using promiscuous 2-keto-aciddecarboxylase and alcohol dehydrogenase (ADH). The 2-keto-aciddecarboxylase activity can be provided by one of the following genes:PDC6 from Saccharomyces cerevisiae, kind from Lactococcus lactis, andTHI3 Saccharomyces cerevisiae (α-ketoisocaproate decarboxylase) and pdcClostridium acetobutylicum. The alcohol dehydrogenase activity can beprovided by ADH2 from Saccharomyces cerevisiae.

Thus, the disclosure provides metabolically-modified photosyntheticmicroorganisms that use CO₂ as a sole carbon source and includerecombinant biochemical pathways useful for producing biofuels such asisobutanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 2-phenylethanol,1-propanol, or 1-butanol via conversion of a suitable substrate by ametabolically engineered microorganism. Also provided are methods ofproducing biofuels using microorganisms described herein.

In one embodiment, a recombinant photosynthetic microorganism thatproduces an alcohol is provided. The alcohol can be 1-propanol,isobutanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol or2-phenylethanol. In general, the alcohol may be produced fermentativelyor non-fermentatively (i.e., with or without the presence of oxygen)from a metabolite comprising 2-keto acid. In some embodiments, the2-keto acid includes 2-ketobutyrate, 2-ketoisovalerate, 2-ketovalerate,2-keto-3-methylvalerate, 2-keto-4-methyl-pentanoate, or phenylpyruvate.In other embodiments, the recombinant microorganism includes elevatedexpression or activity of a 2-keto-acid decarboxylase and an alcoholdehydrogenase, as compared to a parental microorganism. The 2-keto-aciddecarboxylase may be Pdc6 from Saccharomyces cerevisiae, Aro10 fromSaccharomyces cerevisiae, Thi3 from Saccharomyces cerevisiae, Kivd fromLactococcus lactis, or Pdc from Clostridium acetobutylicum, or homologsthereof. The 2-keto-acid decarboxylase can be encoded by a nucleic acidsequence derived from a gene selected from PDC6 from S. cerevisiae,ARO10 from S. cerevisiae, THI3 from S. cerevisiae, kivd from L. lactis,or pdc from C. acetobutylicum, or homologs thereof (See, e.g., SEQ IDNOs: 18-27). In some embodiments, the alcohol dehydrogenase may be Adh2from S. cerevisiae, or homologs thereof (see, e.g., SEQ ID NO:28-29),encoded by a nucleic acid sequence derived from the ADH2 gene from S.cerevisiae. In another embodiment, a recombinant photoautotrophmicroorganism that produces isobutanol is provided. The microorganismincludes elevated expression or activity of acetohydroxy acid synthase,acetohydroxy acid isomeroreductase, dihydroxy-acid dehydratase,2-keto-acid decarboxylase, and alcohol dehydrogenase, as compared to aparental microorganism. As described herein the microorganism is derivedfrom a parental organism that utilizes CO₂ as a sole carbon source(i.e., a photoautotroph).

The amino acid pathways can be amplified by either overexpression oftargeted enzymes and/or by mutagenesis followed by amino acid analogselection. The step from keto acid to alcohols is achieved by expressionof the corresponding proteins in the organism. This process can beachieved either in one organism or multiple organisms. In the lattercase, for example, one organism produces keto acids from CO₂ andexcretes the products to the medium, which are then converted toalcohols by the second organism.

Accordingly, the disclosure also provides a mixed culture comprising aphotoautotroph microorganism and a recombinant photoheterotrophmicroorganism comprising a modified pathway for the production of abiofuel.

In some embodiments, the photoautotroph microorganism or a microorganismcultured with a photoautotroph can include elevated expression ofacetolactate synthase, acetohydroxy acid isomeroreductase,dihydroxy-acid dehydratase, 2-keto-acid decarboxylase, and alcholdehydrogenase. In some embodiments, the recombinant microorganismfurther includes an elevated level of pyruvate as compared to a parentalmicroorganism. The recombinant microorganism may further include thedeletion or inhibition of expression of an adh, ldh, frd, fnr, pflB, orpta gene, or any combination thereof. In particular, the recombinantmicroorganism can include a deletion of adh, ldh, frd alone or incombination with fnr, fnr and pta, or pta and pflB. In one embodiment,the acetohydroxy acid synthase may be encoded by a polynucleotidederived from the ilvIH operon, ilvBN operon, ilvGM in E. coli, or thealsS gene from Bacillus subtilis, or homologs thereof. The ilvI gene ofthe ilvIH operon encodes an acetohydroxyacid synthase large subunitpolypeptide and the ilvH gene of the ilvIH operon encodes anacetohydroxyacid synthase small subunit polypeptide. In anotherembodiment, the acetohydroxy acid isomeroreductase may be encoded by apolynucleotide derived from an ilvC gene in E. coli, or homologsthereof. In another embodiment, the dihydroxy-acid dehydratase may beencoded by a polynucleotide derived from an ilvD gene, or homologsthereof. In yet another aspect, the 2-keto-acid decarboxylase may beencoded by a nucleic acid sequence derived from a kivd gene fromLactococcus lactis or homologs thereof, or an ARO10 gene from S.cerevisiae, or homologs thereof. In a further embodiment, the alcoholdehydrogenase may be encoded by a polynucleotide derived from an ADH2gene from S. cerevisiae, or homologs thereof.

In general the ilvIH operon of Escherichia coli encodes acetohydroxyacid synthase, the first enzyme in the isoleucine, valine and leucinebiosynthetic pathway. The acetohydroxy acid synthase III isozyme, whichcatalyzes the first common step in the biosynthesis of isoleucine,leucine, and valine in Escherichia coli K-12, is composed of twosubunits, the ilvI (acetohydroxyacid synthase III large subunit) andilvH (acetohydroxyacid synthase small subunit) gene products. The ilvCgene of Escherichia coli encodes acetohydroxy acid isomeroreductase, thesecond enzyme in the parallel isoleucine-valine biosynthetic pathway.The ilvD gene of Escherichia coli encodes dihydroxy-acid dehydratase,the third enzyme in the isoleucine-valine biosynthetic pathway. In someembodiments the recombinant microorganism included an elevatedexpression of acetolactate synthase. The acetolactate synthase can beAlsS from Bacillus subtilis.

In one embodiment, a recombinant photoautotroph microorganism orcombination culture comprising an photoautotroph and a recombinantmicroorganism that produces 1-butanol is provided. At least onemicroorganism includes elevated expression or activity of2-isopropylmalate synthase, beta-isopropylmalate dehydrogenase,isopropylmalate isomerase, and threonine dehydratase, as compared to aparental microorganism. In another embodiment, the recombinantmicroorganism further includes increased levels of 2-ketovalerate, ascompared to a parental microorganism. In another embodiment, therecombinant microorganism further includes decreased levels of2-ketoisovalerate, 2-keto-3-methyl-valerate, or2-keto-4-methyl-pentanoate, or any combination thereof, as compared to aparental microorganism. Accordingly, the microorganism may furtherinclude the deletion or inhibition of expression of an ilvD gene, ascompared to a parental microorganism. In one embodiment, the2-isopropylmalate synthase may be encoded by a polynucleotide derivedfrom a leuA gene, or homologs thereof. In another aspect, thebeta-isopropylmalate dehydrogenase may be encoded by a polynucleotidederived from a leuB gene, or homologs thereof. In yet anotherembodiment, the isopropylmalate isomerase may be encoded by apolynucleotide derived from a leuCD operon, or homologs thereof. Ingeneral the leuC gene of the leuCD operon encodes an isopropylmalateisomerase large subunit polypeptide and the leuD gene of the leuCDoperon encodes an isopropylmalate isomerase small subunit polypeptide.In another embodiment, the threonine dehydratase may be encoded by apolynucleotide derived from an ilvA gene, or homologs thereof. In yetanother embodiment, the threonine dehydratase may be encoded by apolynucleotide derived from a tdcB gene, or homologs thereof. In yetanother embodiment, the recombinant microorganism may further includeelevated expression or activity of pyruvate carboxylase, aspartateaminotransferase, homoserine dehydrogenase, aspartate-semialdehydedehydrogenase, homoserine kinase, threonine synthase, L-serinedehydratase, or threonine dehydratase, or any combination thereof, ascompared to a parental microorganism. In some embodiments, the pyruvatecarboxylase, aspartate aminotransferase, homoserine dehydrogenase,aspartate-semialdehyde dehydrogenase, homoserine kinase, threoninesynthase, L-serine dehydratase, and threonine dehydratase, are encodedby a polynucleotide derived from the ppc, pyc, aspC, thrA, asd, thrB,thrC, sdaAB, and tdcB genes, respectively, or homologs thereof.

In one embodiment, a recombinant photoautotroph microorganism orcombination culture comprising an photoautotroph and a recombinantmicroorganism that produces 1-propanol is provided. The microorganismincludes elevated expression or activity of alpha-isopropylmalatesynthase, LeuB of Leptospira interrogans, isopropylmalate isomerase, andthreonine dehydratase, as compared to a parental microorganism. In oneembodiment, the alpha-isopropylmalate synthase may be encoded by apolynucleotide derived from a cimA gene, or homologs thereof. The cimAgene may be a Leptospira interrogans cimA gene or Methanocaldococcusjannaschii cimA gene. In another embodiment, the beta-isopropylmalatedehydrogenase may be encoded by a polynucleotide derived from a leuBgene, or homologs thereof. In another embodiment, the isopropylmalateisomerase may be encoded by a polynucleotide derived from a leuCDoperon, or homologs thereof. In yet another embodiment, the recombinantmicroorganism may further include elevated expression or activity ofphosphoenolpyruvate carboxylase, pyruvate carboxylase, aspartateaminotransferase, homoserine dehydrogenase, aspartate-semialdehydedehydrogenase, homoserine kinase, threonine synthase, L-serinedehydratase, or threonine dehydratase, or any combination thereof, ascompared to a parental microorganism. In some embodiments, the pyruvatecarboxylase, aspartate aminotransferase, homoserine dehydrogenase,aspartate-semialdehyde dehydrogenase, homoserine kinase, threoninesynthase, L-serine dehydratase, and threonine dehydratase, are encodedby a polynucleotide derived from the ppc, pyc, aspC, thrA, asd, thrB,thrC, sdaAB, and tdcB genes, respectively, or homologs thereof.

In another embodiment, a recombinant photoautotroph microorganism orcombination culture comprising an photoautotroph and a recombinantmicroorganism that produces 2-methyl 1-butanol is provided. Themicroorganism includes elevated expression or activity of threoninedehydratase, acetohydroxy acid synthase, acetohydroxy acidisomeroreductase, dihydroxy-acid dehydratase, 2-keto-acid decarboxylase,and alcohol dehydrogenase, as compared to a parental microorganism,wherein the recombinant microorganism produces 2-methyl 1-butanol. Insome embodiments, the threonine dehydratase may be encoded by apolynucleotide derived from an ilvA gene, or homologs thereof. Inanother embodiment, the threonine dehydratase may be encoded by apolynucleotide derived from a tdcB gene, or homologs thereof. In anotherembodiment, the recombinant microorganism further includes increasedlevels of 2-keto-3-methyl-valerate, as compared to a parentalmicroorganism. In yet another embodiment, the 2-keto-acid decarboxylasemay be encoded by a polynucleotide derived from a kivd gene, or homologsthereof, or a PDC6 gene, or homologs thereof, or THI3 gene, or homologsthereof.

In another embodiment, a recombinant photoautotroph microorganism orcombination culture comprising a photoautotroph and a recombinantmicroorganism that produces 3-methyl 1-butanol is provided. Themicroorganism includes elevated expression or activity of acetohydroxyacid synthase or acetolactate synthase, acetohydroxy acidisomeroreductase, dihydroxy-acid dehydratase, 2-isopropylmalatesynthase, isopropylmalate isomerase, beta-isopropylmalate dehydrogenase,2-keto-acid decarboxylase, and alcohol dehydrogenase, as compared to aparental microorganism. In some embodiments, the acetohydroxy acidsynthase may be encoded by a polynucleotide derived from an ilvIHoperon, or homologs thereof. In another embodiment, the acetolactatesynthase may be encoded by a polynucleotide derived from an alsS gene,or homologs thereof. In another embodiment, the acetolactate synthasemay be encoded by a polynucleotide derived from an ilvMG operon, orhomologs thereof. In another embodiment, the recombinant microorganismfurther includes increased levels of 2-ketoisocaproate, as compared to aparental microorganism. In yet another embodiment, the acetolactatesynthase may be encoded by a polynucleotide derived from an ilvNBoperon, or homologs thereof.

In another embodiment, a recombinant photoautotroph microorganism orcombination culture comprising an photoautotroph and a recombinantmicroorganism that produces phenylethanol is provided. The microorganismincludes elevated expression or activity of chorismate mutaseP/prephenate dehydratase, chorismate mutase T/prephenate dehydrogenase,2-keto-acid decarboxylase and alcohol dehydrogenase, as compared to aparental microorganism. In one embodiment, the chorismate mutaseP/prephenate dehydratase may be encoded by a polynucleotide derived froma pheA gene, or homologs thereof. In another embodiment, the chorismatemutase T/prephenate dehydrogenase may be encoded by polynucleotidederived from a tyrA gene, or homologs thereof. In yet anotherembodiment, the recombinant microorganism further includes increasedlevels of phenylpyruvate, as compared to a parental microorganism.

In one embodiment, a method of producing a recombinant photoautotrophmicroorganism or combination culture comprising an photoautotroph and arecombinant microorganism that converts a suitable substrate ormetabolic intermediate to 1-butanol is provided. The method includestransforming a microorganism with one or more recombinantpolynucleotides encoding polypeptides comprising 2-isopropylmalatesynthase activity, beta-isopropylmalate dehydrogenase activity,isopropylmalate isomerase activity, and threonine dehydratase activity.

In another embodiment, a method of producing a recombinantphotoautotroph microorganism or combination culture comprising anphotoautotroph and a recombinant microorganism that converts a suitablesubstrate or metabolic intermediate to isobutanol, is provided. Themethod includes transforming a microorganism with one or morerecombinant nucleic acid sequences encoding polypeptides comprisingacetohydroxy acid synthase activity, acetohydroxy acid isomeroreductaseactivity, dihydroxy-acid dehydratase activity, 2-keto-acid decarboxylaseactivity, and alcohol dehydrogenase activity.

In another embodiment, a method of producing a recombinantphotoautotroph microorganism or combination culture comprising aphotoautotroph and a recombinant microorganism that converts a suitablesubstrate or metabolic intermediate to 1-propanol, is provided. Themethod includes transforming a microorganism with one or morerecombinant nucleic acid sequences encoding polypeptides comprisingalpha-isopropylmalate synthase activity, beta-isopropylmalatedehydrogenase activity, isopropylmalate isomerase activity, andthreonine dehydratase activity.

In one embodiment, a method of producing a recombinant photoautotrophmicroorganism or combination culture comprising an photoautotroph and arecombinant microorganism that converts a suitable substrate ormetabolic intermediate to 2-methyl 1-butanol, is provided. The methodincludes transforming a microorganism with one or more recombinantnucleic acid sequences encoding polypeptides comprising threoninedehydratase activity, acetohydroxy acid synthase activity, acetohydroxyacid isomeroreductase activity, dihydroxy-acid dehydratase activity,2-keto-acid decarboxylase activity, and alcohol dehydrogenase activity.

In another embodiment, a method of producing a recombinantphotoautotroph microorganism or combination culture comprising aphotoautotroph and a recombinant microorganism that converts a suitablesubstrate or metabolic intermediate to 3-methyl 1-butanol, is provided.The method includes transforming a microorganism with one or morerecombinant nucleic acid sequences encoding polypeptides comprisingacetohydroxy acid synthase activity or acetolactate synthase activity,acetohydroxy acid isomeroreductase activity, dihydroxy-acid dehydrataseactivity, 2-isopropylmalate synthase activity, isopropylmalate isomeraseactivity, beta-isopropylmalate dehydrogenase activity, 2-keto-aciddecarboxylase activity, and alcohol dehydrogenase activity.

In another embodiment, a method of producing a recombinantphotoautotroph microorganism or combination culture comprising anphotoautotroph and a recombinant microorganism that converts a suitablesubstrate or metabolic intermediate to phenylethanol, is provided. Themethod includes transforming a microorganism with one or morerecombinant nucleic acid sequences encoding polypeptides comprisingchorismate mutase P/prephenate dehydratase activity, chorismate mutaseT/prephenate dehydrogenase activity, 2-keto-acid decarboxylase activity,and alcohol dehydrogenase activity.

In another embodiment, a method of producing an alcohol, is provided.The method includes providing a recombinant photoautotroph microorganismor a culture comprising an photoautotroph and a recombinantmicroorganism provided herein; culturing the microorganism(s) in thepresence of a suitable substrate or metabolic intermediate and underconditions suitable for the conversion of the substrate to an alcohol;and detecting the production of the alcohol. In various aspects, thealcohol is selected from 1-propanol, isobutanol, 1-butanol, 2-methyl1-butanol, 3-methyl 1-butanol, and 2-phenylethanol. In another aspect,the substrate or metabolic intermediate includes a 2-keto acid, such as2-ketobutyrate, 2-ketoisovalerate, 2-ketovalerate, 2-keto3-methylvalerate, 2-keto 4-methyl-pentanoate, or phenylpyruvate.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages will be apparent from the description anddrawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of this specification, illustrate one or more embodiments of thedisclosure and, together with the detailed description, serve to explainthe principles and implementations of the invention.

FIG. 1A depicts an exemplary synthetic non-fermentative pathway using2-keto acid metabolism for alcohol production.

FIG. 1B depicts an exemplary alcohol production pathway in geneticallyengineered E. coli. Red arrowheads represent the 2-keto acid degradationpathway. Blue enzyme names represent amino acid biosynthesis pathways.Double lines represent a side-reaction of amino acid biosynthesispathways.

FIG. 1C shows an exemplary biosynthetic pathway in photoautotrophs forthe production of isobutylaldehyde.

Figure D-J shows isobutyraldehyde production from cyanobacteria. (D,E)Schematic representation of recombination to integrate kivd (D) alsS,ilvC and ilvD (E) genes into the S. elongatus chromosome. (F) Specificactivities of AlsS, IlvC and IlvD in cell extracts of SA578 (withintegrated kivd only) and SA590 (with integrated kivd, alsS, ilvC andilvD). Detailed methods and unit definitions of enzyme assays aredescribed in Online Methods. Error indicates s.d. (G) Cumulativeproduction of isobutyraldehyde production by SA590. (H) Daily productionrate of isobutyraldehyde by SA590. (I) Isobutyraldehyde concentration inthe production culture of SA590. (J) Time courses for the growth ofSA590. Error bars indicate s.d. (n=3).

Figures K-L shows ADH comparison for isobutanol production K. Comparisonof isobutanol and isobutyraldehyde production by each combination ofoverexpression of ADHs with or without KIV (10 g/L) supplementation. Thecells (SA413, SA561, SA562) were grown in shake flasks at 30° C. with 1mM IPTG for 24 hr. L. Isobutanol production and time courses for thegrowth of SA561 (without alsS-ilvC-ilvD (squares)) and SA579 (withalsS-ilvC-ilvD (circles)).

FIG. 1M-O shows isobutanol production and comparison of variouscyanobacterial and algal productivities. (M) Isobutanol production fromNaHCO3 using SA579 (with integrated alsS, ilvCD, kivd and yqhD) in shakeflasks without stripping. Only trace amounts (<10 mg/l) ofisobutyraldehyde were detected, indicating the dehydrogenase activity ofYqhD was sufficient for isobutanol production. (N) Time course for thegrowth of SA579. Error bars indicate s.d. (n=3). (O) Productivitycomparison of various processes. Productivities (μg l⁻¹ h⁻¹) ofisobutyraldehyde production (this work), isobutanol production, ethanolproduction from S. elongatus1, hydrogen production from (1) Anabaenavariabilis PK84, (2) Anabaena variabilis AVM13, (3) Chlamydomonasreinhardtii31 (4) Oscillatoria sp. Miami BG7, and lipid production fromHaematococcus pluvialis.

FIG. 1P-Q shows S. elongatus tolerance to isobutyraldehyde andisobutanol Effect of isobutyraldehyde (P) or isobutanol (Q) addition ongrowing cultures of S. elongatus as determined by optical density(OD730). At OD730 ˜1.0, isobutyraldehyde or isobutanol was added to thecultures at final concentration (mg/L) of: 0 (squares), 500 (circles),750 (triangles), 1000 (diamonds) and 2500 (stars).

FIG. 2 depicts modified amino acid biosynthesis pathways for improvedisobutanol and 1-butanol production. Panel A shows isobutanol productionwith or without the engineered ilvIHCD pathway. Left panel: isobutanolproduction; Right panel: isobutanol yield per g of glucose. Theoreticalmaximum yield of isobutanol is 0.41 g/g. Panel B shows 1-butanolproduction with or without the engineered ilvA-leuABCD pathway fromglucose. Left panel: 1-butanol production; Right panel: 1-propanolproduction in the same strain. Panel C shows 1-butanol production withL-threonine addition. Left panel: 1-butanol production; Right panel:1-propanol production from the same strain.

FIG. 3 depicts an exemplary pathway for the production of2-keto-isovalerate from pyruvate.

FIG. 4 depicts an exemplary pathway for leucine biosynthesis.

FIG. 5 depicts an exemplary pathway for isoleucine biosynthesis.

FIG. 6 depicts an exemplary pathway for butanol biosynthesis including2-ketobutyrate as a biosynthetic intermediate.

FIG. 7 depicts an exemplary pathway for butanol biosynthesis frompyruvate.

FIG. 8 depicts an exemplary pathway for butanol biosynthesis includingthreonine as a biosynthetic intermediate.

FIG. 9 depicts exemplary biosynthetic pathways for the production ofisobutanol (e.g., 2-methylpropyl alcohol), 3-methyl 1-butanol,1-butanol, ethanol, 2-methyl 1-butanol, and 1-propanol.

FIG. 10 depicts exemplary biosynthetic pathways for the production ofphenylethanol, ethanol, 3-methyl 1-butanol, and isobutanol (e.g.,2-methylpropyl alcohol).

FIG. 11 depicts a nucleic acid sequence derived from a kivd geneencoding a polypeptide having 2-keto-acid decarboxylase activity.

FIG. 12 depicts a nucleic acid sequence derived from a PDC6 geneencoding a polypeptide having 2-keto-acid decarboxylase activity.

FIG. 13 depicts a nucleic acid sequence derived from a ARO10 geneencoding a polypeptide having 2-keto-acid decarboxylase activity.

FIG. 14 depicts a nucleic acid sequence derived from an THI3 geneencoding a polypeptide having 2-keto-acid decarboxylase activity.

FIG. 15 depicts a nucleic acid sequence derived from an pdc geneencoding a polypeptide having 2-keto-acid decarboxylase activity.

FIG. 16 depicts a nucleic acid sequence derived from a ADH2 geneencoding a polypeptide having alcohol dehydrogenase activity.

FIG. 17 depicts a nucleic acid sequence derived from an ilvI geneencoding a polypeptide having acetolactate synthase large subunitactivity.

FIG. 18 depicts a nucleic acid sequence derived from an ilvH geneencoding a polypeptide having acetolactate synthase small subunitactivity.

FIG. 19 depicts a nucleic acid sequence derived from a ilvC geneencoding a polypeptide having acetohydroxy acid isomeroreductaseactivity.

FIG. 20 depicts a nucleic acid sequence derived from a ilvD geneencoding a polypeptide having dihydroxy-acid dehydratase activity.

FIG. 21 depicts a nucleic acid sequence derived from a ilvA geneencoding a polypeptide having threonine dehydratase activity.

FIG. 22 depicts a nucleic acid sequence derived from a leuA geneencoding a polypeptide having 2-isopropylmalate synthase activity.

FIG. 23 depicts a nucleic acid sequence derived from a leuB geneencoding a polypeptide having beta-isopropylmalate dehydrogenaseactivity.

FIG. 24 depicts a nucleic acid sequence derived from a leuC geneencoding a polypeptide having isopropylmalate isomerase large subunitactivity.

FIG. 25 depicts a nucleic acid sequence derived from a leuD geneencoding a polypeptide having isopropylmalate isomerase small subunitactivity.

FIG. 26 depicts a nucleic acid sequence derived from a cimA geneencoding a polypeptide having alpha-isopropylmalate synthase activity.

FIG. 27 depicts a nucleic acid sequence derived from a ilvM geneencoding a polypeptide having acetolactate synthase large subunitactivity.

FIG. 28 depicts a nucleic acid sequence derived from a ilvG geneencoding a polypeptide having acetolactate synthase small subunitactivity.

FIG. 29 depicts a nucleic acid sequence derived from a ilvN geneencoding a polypeptide having acetolactate synthase large subunitactivity.

FIG. 30 depicts a nucleic acid sequence derived from a ilvB geneencoding a polypeptide having acetolactate synthase small subunitactivity.

FIG. 31 depicts a nucleic acid sequence derived from a adhE2 geneencoding a polypeptide having alcohol dehydrogenase activity.

FIG. 32 depicts a nucleic acid sequence derived from a Li-cimA geneencoding a polypeptide having alpha-isopropylmalate synthase activity.

FIG. 33 depicts a nucleic acid sequence derived from a Li-leuC geneencoding a polypeptide having isopropylmalate isomerase large subunitactivity.

FIG. 34 depicts a nucleic acid sequence derived from a Li-leuD geneencoding a polypeptide having isopropylmalate isomerase small subunitactivity.

FIG. 35 depicts a nucleic acid sequence derived from a Li-leuB geneencoding a polypeptide having beta-isopropylmalate dehydrogenaseactivity.

FIG. 36 depicts a nucleic acid sequence derived from a pheA geneencoding a polypeptide having chorismate mutase P/prephenate dehydrataseactivity.

FIG. 37 depicts a nucleic acid sequence derived from a TyrA geneencoding a polypeptide having chorismate mutase T/prephenate dehydrataseactivity.

FIG. 38 depicts a nucleic acid sequence derived from an alsS geneencoding a polypeptide having acetolactate synthase activity.

FIG. 39 depicts an exemplary isobutanol production pathway via pyruvate.

FIG. 40 depicts an exemplary isobutanol production pathway viaL-threonine.

FIG. 40A depicts reactions 1-5 of an exemplary 1-butanol productionpathway via pyruvate.

FIG. 40B depicts reactions 6-7 of an exemplary 1-butanol productionpathway via pyruvate.

FIG. 41 depicts an exemplary 1-propanol production pathway viaL-threonine.

FIG. 42 depicts an exemplary 1-propanol production pathway via pyruvate.

FIG. 43 depicts an exemplary 2-methyl-1-butanol production pathway viaL-threonine.

FIG. 43A depicts reactions 1-8 of an exemplary 3-methyl-1-butanolproduction pathway via pyruvate.

FIG. 43B depicts reaction 9 of an exemplary 3-methyl-1-butanolproduction pathway via pyruvate.

FIG. 44 depicts an exemplary phenyl-ethanol production pathway viachorismate.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a microorganism”includes a plurality of such microorganisms and reference to “thepolypeptide” includes reference to one or more polypeptides andequivalents thereof, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although any methods andreagents similar or equivalent to those described herein can be used inthe practice of the disclosed methods and compositions, the exemplarymethods and materials are now described.

Also, the use of “or” means “and/or” unless stated otherwise. Similarly,“comprise,” “comprises,” “comprising” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

All publications mentioned herein are incorporated herein by referencein full for the purpose of describing and disclosing the methodologies,which are described in the publications, which might be used inconnection with the description herein. The publications discussed aboveand throughout the text are provided solely for their disclosure priorto the filing date of the present application. Nothing herein is to beconstrued as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior disclosure.

The disclosure provides methods and compositions for the production ofhigher alcohols using a culture of microorganisms that utilizes CO₂ as acarbon source. Examples of such microorganisms that utilize CO₂ as acarbon source include photoautotrophs. In some embodiments, that methodsand compositions comprise a co-culture of photoautotrophs and aphotoheterotroph or a photoautotroph and a microorganism that cannotutilize CO₂ as a carbon source.

In various embodiments the metabolically engineered microorganisms orcombination cultures provided herein include biochemical pathways forthe production of high alcohols including isobutanol, 1-butanol,1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol and 2-phenylethanolfrom a suitable substrate. In various embodiments a recombinantmicroorganism provided herein includes the elevated expression orexpression of a heterologous polypeptide of at least one target enzymeas compared to a parental microorganism. The recombinant microorganismalso produces at least one metabolite involved in a biosynthetic pathwayfor the production of isobutanol, 1-butanol, 1-propanol,2-methyl-1-butanol, 3-methyl-1-butanol or 2-phenylethanol. In general,the microorganisms or combination culture provided herein include atleast one recombinant metabolic pathway that includes a target enzyme.The pathway acts to modify a substrate or metabolic intermediate in theproduction of isobutanol, 1-butanol, 1-propanol, 2-methyl-1-butanol,3-methyl-1-butanol or 2-phenylethanol. The target enzyme is encoded by,and expressed from, a nucleic acid sequence derived from a suitablebiological source. In some embodiments the polynucleotide is a genederived from a bacterial or yeast source.

As used herein, the term “metabolically engineered” or “metabolicengineering” involves rational pathway design and assembly ofbiosynthetic genes, genes associated with operons, and control elementsof such nucleic acid sequences, for the production of a desiredmetabolite, such as a 2-keto acid or high alcohol, in a microorganism.“Metabolically engineered” can further include optimization of metabolicflux by regulation and optimization of transcription, translation,protein stability and protein functionality using genetic engineeringand appropriate culture condition. The biosynthetic genes can beheterologous to the host (e.g., microorganism), either by virtue ofbeing foreign to the host, or being modified by mutagenesis,recombination, and/or association with a heterologous expression controlsequence in an endogenous host cell. Appropriate culture conditions areconditions of culture medium pH, ionic strength, nutritive content,etc.; temperature; oxygen/CO₂/nitrogen content; humidity; and otherculture conditions that permit production of the compound by the hostmicroorganism, i.e., by the metabolic action of the microorganism.Appropriate culture conditions are well known for microorganisms thatcan serve as host cells.

Accordingly, metabolically “engineered” or “modified” microorganisms areproduced via the introduction of genetic material into a host orparental microorganism of choice thereby modifying or altering thecellular physiology and biochemistry of the microorganism. Through theintroduction of genetic material the parental microorganism acquires newproperties, e.g. the ability to produce a new, or greater quantities of,an intracellular metabolite. In an illustrative embodiment, theintroduction of genetic material into a parental microorganism resultsin a new or modified ability to produce an alcohol such as 1-propanol,isobutanol, 1-butanol, 2-methyl 1-butanol, 3-methyl 1-butanol or2-phenylethanol. The genetic material introduced into the parentalmicroorganism contains gene(s), or parts of genes, coding for one ormore of the enzymes involved in a biosynthetic pathway for theproduction of an alcohol and may also include additional elements forthe expression and/or regulation of expression of these genes, e.g.promoter sequences.

Microorganisms provided herein are modified to produce metabolites inquantities not available in the parental microorganism. A “metabolite”refers to any substance produced by metabolism or a substance necessaryfor or taking part in a particular metabolic process. A metabolite canbe an organic compound that is a starting material (e.g., glucose orpyruvate) in, an intermediate (e.g., 2-keto acid) in, or an end product(e.g., 1-propanol, isobutanol, 1-butanol, 2-methyl 1-butanol, 3-methyl1-butanol or 2-phenylethanol) of metabolism. Metabolites can be used toconstruct more complex molecules, or they can be broken down intosimpler ones. Intermediate metabolites may be synthesized from othermetabolites, perhaps used to make more complex substances, or brokendown into simpler compounds, often with the release of chemical energy.End products of metabolism are the final result of the breakdown ofother metabolites.

FIG. 1A shows a general pathway for production of a biofuel in arecombinant microorganism or co-culture of the disclosure from CO₂ as acarbon source. Exemplary metabolites include glucose, pyruvate,1-propanol, isobutanol, 1-butanol, 2-methyl 1-butanol, 3-methyl1-butanol or 2-phenylethanol, and 2-keto acids. As depicted in FIG. 1B,exemplary 2-keto acids include 2-ketobutyrate, 2-ketoisovalerate,2-ketovalerate, 2-keto 3-methylvalerate, 2-keto 4-methyl-pentanoate, andphenylpyruvate. The exemplary 2-keto acids shown in FIG. 1B may be usedas metabolic intermediates in the production of 1-propanol, isobutanol,1-butanol, 2-methyl 1-butanol, 3-methyl 1-butanol or 2-phenylethanol.For example, as shown in FIG. 1B a recombinant microorganismmetabolically engineered to provide elevated expression of enzymesencoded by LeuABCD produces 2-ketovalerate from 2-ketobutyrate. The2-ketovalerate metabolite may be used to produce 1-butanol by additionalenzymes produced by the metabolically modified microorganism.Additionally, 1-propanol and 2-methyl 1-butanol can be produced from2-ketobutyrate and 2-keto-3-methyl-valerate by a recombinantmicroorganism metabolically engineered to express or over-expressenzymes encoded by ilvIHDC, KDC and ADH genes. Further, the metabolite2-ketoisovalerate can be produced by a recombinant microorganismmetabolically engineered to express or over-express enzymes encoded byilvIHCD genes. This metabolite can then be used in the production ofisobutanol or 3-methyl 1-butanol. The metabolites pyruvate andphenylpyruvate can be used to produce 2-phenylethanol by a recombinantmicroorganism metabolically engineered to express or over-expressenzymes encoded by KDC and ADH. Additional metabolites and genes areshown in FIG. 1B.

The term “biosynthetic pathway”, also referred to as “metabolicpathway”, refers to a set of anabolic or catabolic biochemical reactionsfor converting (transmuting) one chemical species into another. Geneproducts belong to the same “metabolic pathway” if they, in parallel orin series, act on the same substrate, produce the same product, or acton or produce a metabolic intermediate (i.e., metabolite) between thesame substrate and metabolite end product.

The term “substrate” or “suitable substrate” refers to any substance orcompound that is converted or meant to be converted into anothercompound by the action of an enzyme. The term includes not only a singlecompound, but also combinations of compounds, such as solutions,mixtures and other materials which contain at least one substrate, orderivatives thereof. Further, the term “substrate” encompasses not onlycompounds that provide a carbon source suitable for use as a startingmaterial, such as any biomass derived sugar, but also intermediate andend product metabolites used in a pathway associated with ametabolically engineered microorganism as described herein. A “biomassderived sugar” includes, but is not limited to, molecules such asglucose, mannose, xylose, and arabinose or sugars or intermediatesproduced by a photosynthetic microorganism. The term biomass derivedsugar encompasses suitable carbon substrates ordinarily used bymicroorganisms, such as 6 carbon sugars, including but not limited togulose, lactose, sorbose, fructose, idose, galactose and mannose all ineither D or L form, or a combination of 6 carbon sugars, such as glucoseand fructose, and/or 6 carbon sugar acids including, but not limited to,2-keto-L-gulonic acid, idonic acid (IA), gluconic acid (GA),6-phosphogluconate, 2-keto-D-gluconic acid (2 KDG), 5-keto-D-gluconicacid, 2-ketogluconatephosphate, 2,5-diketo-L-gulonic acid,2,3-L-diketogulonic acid, dehydroascorbic acid, erythorbic acid (EA) andD-mannonic acid.

The term “alcohol” includes for example 1-propanol, isobutanol,1-butanol, 2-methyl 1-butanol, 3-methyl 1-butanol or 2-phenylethanol.The term “1-butanol” generally refers to a straight chain isomer withthe alcohol functional group at the terminal carbon. The straight chainisomer with the alcohol at an internal carbon is sec-butanol or2-butanol. The branched isomer with the alcohol at a terminal carbon isisobutanol, and the branched isomer with the alcohol at the internalcarbon is tert-butanol.

Recombinant microorganisms provided herein can express a plurality oftarget enzymes involved in pathways for the production of e.g.,1-propanol, isobutanol, 1-butanol, 2-methyl 1-butanol, 3-methyl1-butanol or 2-phenylethanol, from using a suitable carbon substrate.

Accordingly, provided herein are recombinant microorganisms that produceisobutanol and in some embodiments may include the elevated expressionof target enzymes such as acetohydroxy acid synthase (ilvIH operon),acetohydroxy acid isomeroreductase (ilvC), dihydroxy-acid dehydratase(ilvD), 2-keto-acid decarboxylase (PDC6, ARO10, THI3, kivd, or pdc), andalcohol dehydrogenase (ADH2). The microorganism may further include thedeletion or inhibition of expression of an adh (e.g., an adhE), ldh(e.g., an ldhA), frd (e.g., an frdB, an frdC or an frdBC), fnr, pflB, orpta gene, or any combination thereof, to increase the availability ofpyruvate. In some embodiments the recombinant microorganism may includethe elevated expression of acetolactate synthase (alsS), acteohydroxyacid isomeroreductase (ilvC), dihydroxy-acid dehydratase (ilvD), 2-ketoacid decarboxylase (PDC6, ARO10, TH13, kivd, or pdc), and alcoholdehydrogenase (ADH2). In one embodiment, the recombinant microorganismis an autophototroph or may be a non-photosynthetic organismrecombinantly engineered to produce the alcohol that is cultured incombination with a autophototroph to fix CO₂.

Also provided are recombinant microorganisms that produce 1-butanol andmay include the elevated expression of target enzymes such as2-isopropylmalate synthase (leuA), beta-isopropylmalate dehydrogenase(leuB), isopropylmalate isomerase (leuCD operon), threonine dehydratase(ilvA). The microorganism may be a autophotroph microorganism or anon-photosynthetic or heterotrophic microorganism. The microorganism mayfurther include decreased levels of 2-ketoisovalerate,2-keto-3-methyl-valerate, or 2-keto-4-methyl-pentanoate, or anycombination thereof, as compared to a parental microorganism. Inaddition, the microorganism may include the deletion or inhibition ofexpression of an ilvD gene, as compared to a parental microorganism. Arecombinant microorganism that produces 1-butanol and may includefurther elevated expression or activity of pyruvate carboxylase,aspartate aminotransferase, homoserine dehydrogenase,aspartate-semialdehyde dehydrogenase, homoserine kinase, threoninesynthase, L-serine dehydratase, and/or threonine dehydratase, encoded bya nucleic acid sequences derived from the ppc, pyc, aspC, thrA, asd,thrB, thrC, sdaAB, and tdcB genes, respectively.

Also provided are recombinant microorganisms that produce 1-propanol andmay include the elevated expression of target enzymes such asalpha-isopropylmalate synthase (cimA), beta-isopropylmalatedehydrogenase (leuB), isopropylmalate isomerase (leuCD operon) andthreonine dehydratase.

Also provided are recombinant microorganisms that produce 2-methyl1-butanol and may include the elevated expression of target enzymes suchas threonine dehydratase (ilvA or tdcB), acetohydroxy acid synthase(ilvIH operon), acetohydroxy acid isomeroreductase (ilvC),dihydroxy-acid dehydratase (ilvD), 2-keto-acid decarboxylase (PDC6,ARO10, THI3, kivd, and/or pdc, and alcohol dehydrogenase (ADH2).

Also provided are recombinant photoautotroph microorganism(s) or culturecomprising a photoautotroph and a recombinant non-photosynthetic orphotoheterotroph microorganism that produce 3-methyl 1-butanol and mayinclude the elevated expression of target enzymes such as acetolactatesynthase (alsS), acetohydroxy acid synthase (ilvIH), acetolactatesynthase (ilvMG) or (ilvNB), acetohydroxy acid isomeroreductase (ilvC),dihydroxy-acid dehydratase (ilvD), 2-isopropylmalate synthase (leuA),isopropylmalate isomerase (leuCD operon), beta-isopropylmalatedehydrogenase (leuB), 2-keto-acid decarboxylase (kivd, PDC6, or THI3),and alcohol dehydrogenase (ADH2).

Also provided are recombinant photoautotroph microorganism(s) or culturecomprising a photoautotroph and a recombinant non-photosynthetic orphotoheterotroph microorganism that produce phenylethanol and mayinclude the elevated expression of target enzymes such as chorismatemutase P/prephenate dehydratase (pheA), chorismate mutase T/prephenatedehydrogenase (tyrA), 2-keto-acid decarboxylase (kivd, PDC6, or THI3),and alcohol dehydrogenase (ADH2).

As previously noted the target enzymes described throughout thisdisclosure generally produce metabolites. For example, the enzymes2-isopropylmalate synthase (leuA), beta-isopropylmalate dehydrogenase(leuB), and isopropylmalate isomerase (leuCD operon) may produce2-ketovalerate from a substrate that includes 2-ketobutyrate. Inaddition, the target enzymes described throughout this disclosure areencoded by nucleic acid sequences. For example, threonine dehydratasecan be encoded by a nucleic acid sequence derived from an ilvA gene.Acetohydroxy acid synthase can be encoded by a nucleic acid sequencederived from an ilvIH operon. Acetohydroxy acid isomeroreductase can beencoded by a nucleic acid sequence derived from an ilvC gene.Dihydroxy-acid dehydratase can be encoded by a nucleic acid sequencederived from an ilvD gene. 2-keto-acid decarboxylase can be encoded by anucleic acid sequence derived from a PDC6, ARO10, THI3, kivd, and/or pdcgene. Alcohol dehydrogenase can be encoded by a nucleic acid sequencederived from an ADH2 gene. Additional enzymes and exemplary genes aredescribed throughout this document. Homologs of the various polypeptidesand nucleic acid sequences can be derived from any biologic source thatprovides a suitable nucleic acid sequence encoding a suitable enzyme.

It is understood that a range of microorganisms can be modified toinclude a recombinant metabolic pathway suitable for the production ofe.g., 1-propanol, isobutanol, 1-butanol, 2-methyl 1-butanol, 3-methyl1-butanol or 2-phenylethanol. It is also understood that variousmicroorganisms can act as “sources” for genetic material encoding targetenzymes suitable for use in a recombinant microorganism provided herein.The term “microorganism” includes prokaryotic and eukaryoticphotosynthetic microbial species. The terms “microbial cells” and“microbes” are used interchangeably with the term microorganism.

“Bacteria”, or “eubacteria”, refers to a domain of prokaryoticorganisms. Bacteria include at least 11 distinct groups as follows: (1)Gram-positive (gram+) bacteria, of which there are two majorsubdivisions: (1) high G+C group (Actinomycetes, Mycobacteria,Micrococcus, others) (2) low G+C group (Bacillus, Clostridia,Lactobacillus, Staphylococci, Streptococci, Mycoplasmas); (2)Proteobacteria, e.g., Purple photosynthetic+non-photosyntheticGram-negative bacteria (includes most “common” Gram-negative bacteria);(3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes andrelated species; (5) Planctomyces; (6) Bacteroides, Flavobacteria; (7)Chlamydia; (8) Green sulfur bacteria; (9) Green non-sulfur bacteria(also anaerobic phototrophs); (10) Radioresistant micrococci andrelatives; (11) Thermotoga and Thermosipho thermophiles.

“Gram-negative bacteria” include cocci, nonenteric rods, and entericrods. The genera of Gram-negative bacteria include, for example,Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella,Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella,Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter,Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium,Chlamydia, Rickettsia, Treponema, and Fusobacterium.

“Gram positive bacteria” include cocci, nonsporulating rods, andsporulating rods. The genera of gram positive bacteria include, forexample, Actinomyces, Bacillus, Clostridium, Corynebacterium,Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus,Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

Photoautotrophic bacteria are typically Gram-negative rods which obtaintheir energy from sunlight through the processes of photosynthesis. Inthis process, sunlight energy is used in the synthesis of carbohydrates,which in recombinant photoautotrophs can be further used asintermediates in the synthesis of biofuels. In other embodiment, thephotoautotrophs serve as a source of carbohydrates for use bynon-photosynthetic microorganism (e.g., recombinant E. coli) to producebiofuels by a metabolically engineered microorganism. Certainphotoautotrophs called anoxygenic photoautotrophs grow only underanaerobic conditions and neither use water as a source of hydrogen norproduce oxygen from photosynthesis. Other photoautotrophic bacteria areoxygenic photoautotrophs. These bacteria are typically cyanobacteria.They use chlorophyll pigments and photosynthesis in photosyntheticprocesses resembling those in algae and complex plants. During theprocess, they use water as a source of hydrogen and produce oxygen as aproduct of photosynthesis.

Cyanobacteria include various types of bacterial rods and cocci, as wellas certain filamentous forms. The cells contain thylakoids, which arecytoplasmic, platelike membranes containing chlorophyll. The organismsproduce heterocysts, which are specialized cells believed to function inthe fixation of nitrogen compounds.

The term “recombinant microorganism” and “recombinant host cell” areused interchangeably herein and refer to microorganisms that have beengenetically modified to express or over-express endogenous nucleic acidsequences, or to express non-endogenous sequences, such as thoseincluded in a vector. The nucleic acid sequence generally encodes atarget enzyme involved in a metabolic pathway for producing a desiredmetabolite as described above. Accordingly, recombinant microorganismsdescribed herein have been genetically engineered to express orover-express target enzymes not previously expressed or over-expressedby a parental microorganism. It is understood that the terms“recombinant microorganism” and “recombinant host cell” refer not onlyto the particular recombinant microorganism but to the progeny orpotential progeny of such a microorganism.

A “parental microorganism” refers to a cell used to generate arecombinant microorganism. The term “parental microorganism” describes acell that occurs in nature, i.e. a “wild-type” cell that has not beengenetically modified. The term “parental microorganism” also describes acell that has been genetically modified but which does not express orover-express a target enzyme e.g., an enzyme involved in thebiosynthetic pathway for the production of a desired metabolite such as1-propanol, isobutanol, 1-butanol, 2-methyl 1-butanol, 3-methyl1-butanol or 2-phenylethanol. For example, a wild-type microorganism canbe genetically modified to express or over express a first target enzymesuch as thiolase. This microorganism can act as a parental microorganismin the generation of a microorganism modified to express or over-expressa second target enzyme e.g., hydroxybutyryl CoA dehydrogenase. In turn,the microorganism modified to express or over express e.g., thiolase andhydroxybutyryl CoA dehydrogenase can be modified to express or overexpress a third target enzyme e.g., crotonase. Accordingly, a parentalmicroorganism functions as a reference cell for successive geneticmodification events. Each modification event can be accomplished byintroducing a nucleic acid molecule in to the reference cell. Theintroduction facilitates the expression or over-expression of a targetenzyme. It is understood that the term “facilitates” encompasses theactivation of endogenous nucleic acid sequences encoding a target enzymethrough genetic modification of e.g., a promoter sequence in a parentalmicroorganism. It is further understood that the term “facilitates”encompasses the introduction of exogenous nucleic acid sequencesencoding a target enzyme in to a parental microorganism.

In another embodiment a method of producing a recombinant microorganismthat converts a suitable carbon substrate to e.g., 1-propanol,isobutanol, 1-butanol, 2-methyl 1-butanol, 3-methyl 1-butanol or2-phenylethanol is provided. The method includes transforming amicroorganism with one or more recombinant nucleic acid sequencesencoding polypeptides that include e.g., acetohydroxy acid synthase(ilvIH operon), acetohydroxy acid isomeroreductase (ilvC),dihydroxy-acid dehydratase (ilvD), 2-keto-acid decarboxylase (PDC6,ARO10, THI3, kivd, or pdc), 2-isopropylmalate synthase (leuA),beta-isopropylmalate dehydrogenase (leuB), isopropylmalate isomerase(leuCD operon), threonine dehydratase (ilvA), alpha-isopropylmalatesynthase (cimA), beta-isopropylmalate dehydrogenase (leuB),isopropylmalate isomerase (leuCD operon), threonine dehydratase (ilvA),acetolactate synthase (ilvMG or ilvNB), acetohydroxy acidisomeroreductase (ilvC), dihydroxy-acid dehydratase (ilvD),beta-isopropylmalate dehydrogenase (leuB), chorismate mutaseP/prephenate dehydratase (pheA), chorismate mutase T/prephenatedehydrogenase (tyrA), 2-keto-acid decarboxylase (kivd, PDC6, or THI3),and alcohol dehydrogenase activity. Nucleic acid sequences that encodeenzymes useful for generating metabolites including homologs, variants,fragments, related fusion proteins, or functional equivalents thereof,are used in recombinant nucleic acid molecules that direct theexpression of such polypeptides in appropriate host cells, such asbacterial or yeast cells. It is understood that the addition ofsequences which do not alter the encoded activity of a nucleic acidmolecule, such as the addition of a non-functional or non-codingsequence, is a conservative variation of the basic nucleic acid. The“activity” of an enzyme is a measure of its ability to catalyze areaction resulting in a metabolite, i.e., to “function”, and may beexpressed as the rate at which the metabolite of the reaction isproduced. For example, enzyme activity can be represented as the amountof metabolite produced per unit of time or per unit of enzyme (e.g.,concentration or weight), or in terms of affinity or dissociationconstants.

A “protein” or “polypeptide”, which terms are used interchangeablyherein, comprises one or more chains of chemical building blocks calledamino acids that are linked together by chemical bonds called peptidebonds. An “enzyme” means any substance, composed wholly or largely ofprotein, that catalyzes or promotes, more or less specifically, one ormore chemical or biochemical reactions. The term “enzyme” can also referto a catalytic polynucleotide (e.g., RNA or DNA). A “native” or“wild-type” protein, enzyme, polynucleotide, gene, or cell, means aprotein, enzyme, polynucleotide, gene, or cell that occurs in nature.

Accordingly, homologs of enzymes useful for generating metabolites(e.g., keto thiolase, acetyl-CoA acetyltransferase, hydroxybutyryl CoAdehydrogenase, crotonase, crotonyl-CoA reductase, butyryl-coAdehydrogenase, alcohol dehydrogenase (ADH)) are encompassed by themicroorganisms and methods provided herein. The term “homologs” usedwith respect to an original enzyme or gene of a first family or speciesrefers to distinct enzymes or genes of a second family or species whichare determined by functional, structural or genomic analyses to be anenzyme or gene of the second family or species which corresponds to theoriginal enzyme or gene of the first family or species. Most often,homologs will have functional, structural or genomic similarities.Techniques are known by which homologs of an enzyme or gene can readilybe cloned using genetic probes and PCR. Identity of cloned sequences ashomolog can be confirmed using functional assays and/or by genomicmapping of the genes.

A protein has “homology” or is “homologous” to a second protein if thenucleic acid sequence that encodes the protein has a similar sequence tothe nucleic acid sequence that encodes the second protein.Alternatively, a protein has homology to a second protein if the twoproteins have “similar” amino acid sequences. (Thus, the term“homologous proteins” is defined to mean that the two proteins havesimilar amino acid sequences).

As used herein, two proteins (or a region of the proteins) aresubstantially homologous when the amino acid sequences have at leastabout 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identity. To determine the percent identity of twoamino acid sequences, or of two nucleic acid sequences, the sequencesare aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second amino acid or nucleicacid sequence for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes). In one embodiment, the length of areference sequence aligned for comparison purposes is at least 30%,typically at least 40%, more typically at least 50%, even more typicallyat least 60%, and even more typically at least 70%, 80%, 90%, 100% ofthe length of the reference sequence. The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

When “homologous” is used in reference to proteins or peptides, it isrecognized that residue positions that are not identical often differ byconservative amino acid substitutions. A “conservative amino acidsubstitution” is one in which an amino acid residue is substituted byanother amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of homology may be adjusted upwardsto correct for the conservative nature of the substitution. Means formaking this adjustment are well known to those of skill in the art (see,e.g., Pearson et al., 1994, hereby incorporated herein by reference).

The following six groups each contain amino acids that are conservativesubstitutions for one another: 1) Serine (S), Threonine (T); 2) AsparticAcid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4)Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine(M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W).

Sequence homology for polypeptides, which is also referred to as percentsequence identity, is typically measured using sequence analysissoftware. See, e.g., the Sequence Analysis Software Package of theGenetics Computer Group (GCG), University of Wisconsin BiotechnologyCenter, 910 University Avenue, Madison, Wis. 53705. Protein analysissoftware matches similar sequences using measure of homology assigned tovarious substitutions, deletions and other modifications, includingconservative amino acid substitutions. For instance, GCG containsprograms such as “Gap” and “Bestfit” which can be used with defaultparameters to determine sequence homology or sequence identity betweenclosely related polypeptides, such as homologous polypeptides fromdifferent species of organisms or between a wild type protein and amutein thereof. See, e.g., GCG Version 6.1.

A typical algorithm when comparing a inhibitory molecule sequence to adatabase containing a large number of sequences from different organismsis the computer program BLAST (Altschul, 1990; Gish, 1993; Madden, 1996;Altschul, 1997; Zhang, 1997), especially blastp or tblastn (Altschul,1997). Typical parameters for BLASTp are: Expectation value: (default);Filter: seg (default); Cost to open a gap: 11 (default); Cost to extenda gap: 1 (default); Max. alignments: 100 (default); Word size: 11(default); No. of descriptions: 100 (default); Penalty Matrix:BLOWSUM62.

When searching a database containing sequences from a large number ofdifferent organisms, it is typical to compare amino acid sequences.Database searching using amino acid sequences can be measured byalgorithms other than blastp known in the art. For instance, polypeptidesequences can be compared using FASTA, a program in GCG Version 6.1.FASTA provides alignments and percent sequence identity of the regionsof the best overlap between the query and search sequences (Pearson,1990, hereby incorporated herein by reference). For example, percentsequence identity between amino acid sequences can be determined usingFASTA with its default parameters (a word size of 2 and the PAM250scoring matrix), as provided in GCG Version 6.1, hereby incorporatedherein by reference.

It is understood that the nucleic acid sequences described above include“genes” and that the nucleic acid molecules described above include“vectors” or “plasmids.” For example, a nucleic acid sequence encoding aketo thiolase can be encoded by an atoB gene or homolog thereof, or anfadA gene or homolog thereof. Accordingly, the term “gene”, also calleda “structural gene” refers to a nucleic acid sequence that codes for aparticular sequence of amino acids, which comprise all or part of one ormore proteins or enzymes, and may include regulatory (non-transcribed)DNA sequences, such as promoter sequences, which determine for examplethe conditions under which the gene is expressed. The transcribed regionof the gene may include untranslated regions, including introns,5′-untranslated region (UTR), and 3′-UTR, as well as the codingsequence. The term “nucleic acid” or “recombinant nucleic acid” refersto polynucleotides such as deoxyribonucleic acid (DNA), and, whereappropriate, ribonucleic acid (RNA). The term “expression” with respectto a gene sequence refers to transcription of the gene and, asappropriate, translation of the resulting mRNA transcript to a protein.Thus, as will be clear from the context, expression of a protein resultsfrom transcription and translation of the open reading frame sequence.

The term “operon” refers two or more genes which are transcribed as asingle transcriptional unit from a common promoter. In some embodiments,the genes comprising the operon are contiguous genes. It is understoodthat transcription of an entire operon can be modified (i.e., increased,decreased, or eliminated) by modifying the common promoter.Alternatively, any gene or combination of genes in an operon can bemodified to alter the function or activity of the encoded polypeptide.The modification can result in an increase in the activity of theencoded polypeptide. Further, the modification can impart new activitieson the encoded polypeptide. Exemplary new activities include the use ofalternative substrates and/or the ability to function in alternativeenvironmental conditions.

A “vector” is any means by which a nucleic acid can be propagated and/ortransferred between organisms, cells, or cellular components. Vectorsinclude viruses, bacteriophage, pro-viruses, plasmids, phagemids,transposons, and artificial chromosomes such as YACs (yeast artificialchromosomes), BACs (bacterial artificial chromosomes), and PLACs (plantartificial chromosomes), and the like, that are “episomes,” that is,that replicate autonomously or can integrate into a chromosome of a hostcell. A vector can also be a naked RNA polynucleotide, a naked DNApolynucleotide, a polynucleotide composed of both DNA and RNA within thesame strand, a poly-lysine-conjugated DNA or RNA, a peptide-conjugatedDNA or RNA, a liposome-conjugated DNA, or the like, that are notepisomal in nature, or it can be an organism which comprises one or moreof the above polynucleotide constructs such as an agrobacterium or abacterium.

“Transformation” refers to the process by which a vector is introducedinto a host cell. Transformation (or transduction, or transfection), canbe achieved by any one of a number of means including electroporation,microinjection, biolistics (or particle bombardment-mediated delivery),or agrobacterium mediated transformation.

Those of skill in the art will recognize that, due to the degeneratenature of the genetic code, a variety of DNA compounds differing intheir nucleotide sequences can be used to encode a given amino acidsequence of the disclosure. The native DNA sequence encoding thebiosynthetic enzymes described above are referenced herein merely toillustrate an embodiment of the disclosure, and the disclosure includesDNA compounds of any sequence that encode the amino acid sequences ofthe polypeptides and proteins of the enzymes utilized in the methods ofthe disclosure. In similar fashion, a polypeptide can typically tolerateone or more amino acid substitutions, deletions, and insertions in itsamino acid sequence without loss or significant loss of a desiredactivity. The disclosure includes such polypeptides with alternate aminoacid sequences, and the amino acid sequences encoded by the DNAsequences shown herein merely illustrate embodiments of the disclosure.

The disclosure provides nucleic acid molecules in the form ofrecombinant DNA expression vectors or plasmids, as described in moredetail below, that encode one or more target enzymes. Generally, suchvectors can either replicate in the cytoplasm of the host microorganismor integrate into the chromosomal DNA of the host microorganism. Ineither case, the vector can be a stable vector (i.e., the vector remainspresent over many cell divisions, even if only with selective pressure)or a transient vector (i.e., the vector is gradually lost by hostmicroorganisms with increasing numbers of cell divisions). Thedisclosure provides DNA molecules in isolated (i.e., not pure, butexisting in a preparation in an abundance and/or concentration not foundin nature) and purified (i.e., substantially free of contaminatingmaterials or substantially free of materials with which thecorresponding DNA would be found in nature) forms.

Provided herein are methods for the heterologous expression of one ormore of the biosynthetic genes involved in 1-propanol, isobutanol,1-butanol, 2-methyl 1-butanol, 3-methyl 1-butanol, and/or2-phenylethanol biosynthesis and recombinant DNA expression vectorsuseful in the method. Thus, included within the scope of the disclosureare recombinant expression vectors that include such nucleic acids. Theterm expression vector refers to a nucleic acid that can be introducedinto a host microorganism or cell-free transcription and translationsystem. An expression vector can be maintained permanently ortransiently in a microorganism, whether as part of the chromosomal orother DNA in the microorganism or in any cellular compartment, such as areplicating vector in the cytoplasm. An expression vector also comprisesa promoter that drives expression of an RNA, which typically istranslated into a polypeptide in the microorganism or cell extract. Forefficient translation of RNA into protein, the expression vector alsotypically contains a ribosome-binding site sequence positioned upstreamof the start codon of the coding sequence of the gene to be expressed.Other elements, such as enhancers, secretion signal sequences,transcription termination sequences, and one or more marker genes bywhich host microorganisms containing the vector can be identified and/orselected, may also be present in an expression vector. Selectablemarkers, i.e., genes that confer antibiotic resistance or sensitivity,are used and confer a selectable phenotype on transformed cells when thecells are grown in an appropriate selective medium.

The various components of an expression vector can vary widely,depending on the intended use of the vector and the host cell(s) inwhich the vector is intended to replicate or drive expression.Expression vector components suitable for the expression of genes andmaintenance of vectors in E. coli, yeast, Streptomyces, and othercommonly used cells are widely known and commercially available. Forexample, suitable promoters for inclusion in the expression vectors ofthe disclosure include those that function in eukaryotic or prokaryotichost microorganisms. Promoters can comprise regulatory sequences thatallow for regulation of expression relative to the growth of the hostmicroorganism or that cause the expression of a gene to be turned on oroff in response to a chemical or physical stimulus. For E. coli andcertain other bacterial host cells, promoters derived from genes forbiosynthetic enzymes, antibiotic-resistance conferring enzymes, andphage proteins can be used and include, for example, the galactose,lactose (lac), maltose, tryptophan (trp), beta-lactamase (bla),bacteriophage lambda PL, and T5 promoters. In addition, syntheticpromoters, such as the tac promoter (U.S. Pat. No. 4,551,433), can alsobe used. For E. coli expression vectors, it is useful to include an E.coli origin of replication, such as from pUC, p1P, p1, and pBR.

Thus, recombinant expression vectors contain at least one expressionsystem, which, in turn, is composed of at least a portion of PKS and/orother biosynthetic gene coding sequences operably linked to a promoterand optionally termination sequences that operate to effect expressionof the coding sequence in compatible host cells. The host cells aremodified by transformation with the recombinant DNA expression vectorsof the disclosure to contain the expression system sequences either asextrachromosomal elements or integrated into the chromosome.

Due to the inherent degeneracy of the genetic code, other nucleic acidsequences which encode substantially the same or a functionallyequivalent amino acid sequence can also be used to clone and express thepolynucleotides encoding such enzymes. As previously noted, the term“host cell” is used interchangeably with the term “recombinantmicroorganism” and includes any cell type which is suitable forproducing e.g., 1-propanol, isobutanol, 1-butanol, 2-methyl 1-butanol,3-methyl 1-butanol and/or 2-phenylethanol and susceptible totransformation with a nucleic acid construct such as a vector orplasmid.

As will be understood by those of skill in the art, it can beadvantageous to modify a coding sequence to enhance its expression in aparticular host. The genetic code is redundant with 64 possible codons,but most organisms typically use a subset of these codons. The codonsthat are utilized most often in a species are called optimal codons, andthose not utilized very often are classified as rare or low-usagecodons. Codons can be substituted to reflect the preferred codon usageof the host, a process sometimes called “codon optimization” or“controlling for species codon bias.”

Optimized coding sequences containing codons preferred by a particularprokaryotic or eukaryotic host (see also, Murray et al. (1989) Nucl.Acids Res. 17:477-508) can be prepared, for example, to increase therate of translation or to produce recombinant RNA transcripts havingdesirable properties, such as a longer half-life, as compared withtranscripts produced from a non-optimized sequence. Translation stopcodons can also be modified to reflect host preference. For example,typical stop codons for S. cerevisiae and mammals are UAA and UGA,respectively. The typical stop codon for monocotyledonous plants is UGA,whereas insects and E. coli commonly use UAA as the stop codon (Dalphinet al. (1996) Nucl. Acids Res. 24: 216-218). Methodology for optimizinga nucleotide sequence for expression in a plant is provided, forexample, in U.S. Pat. No. 6,015,891, and the references cited therein.

A nucleic acid of the disclosure can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques and those procedures described in the Examples section below.The nucleic acid so amplified can be cloned into an appropriate vectorand characterized by DNA sequence analysis. Furthermore,oligonucleotides corresponding to nucleotide sequences can be preparedby standard synthetic techniques, e.g., using an automated DNAsynthesizer.

It is also understood that an isolated nucleic acid molecule encoding apolypeptide homologous to the enzymes described herein can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence encoding the particular polypeptide, suchthat one or more amino acid substitutions, additions or deletions areintroduced into the encoded protein. Mutations can be introduced intothe nucleic acid sequence by standard techniques, such as site-directedmutagenesis and PCR-mediated mutagenesis. In contrast to those positionswhere it may be desirable to make a non-conservative amino acidsubstitutions (see above), in some positions it is preferable to makeconservative amino acid substitutions. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

In another embodiment a method for producing e.g., 1-propanol,isobutanol, 1-butanol, 2-methyl 1-butanol, 3-methyl 1-butanol or2-phenylethanol is provided. The method includes culturing a recombinantphotoautotroph microorganism(s) or culture comprising a photoautotrophand a recombinant non-photosynthetic or photoheterotroph microorganismas provided herein in the presence of a suitable substrate (e.g., CO₂)and under conditions suitable for the conversion of the substrate to1-propanol, isobutanol, 1-butanol, 2-methyl 1-butanol, 3-methyl1-butanol or 2-phenylethanol. The alcohol produced by a microorganism orculture provided herein can be detected by any method known to theskilled artisan. Culture conditions suitable for the growth andmaintenance of a recombinant microorganism provided herein are describedin the Examples below. The skilled artisan will recognize that suchconditions can be modified to accommodate the requirements of eachmicroorganism.

As previously discussed, general texts which describe molecularbiological techniques useful herein, including the use of vectors,promoters and many other relevant topics, include Berger and Kimmel,Guide to Molecular Cloning Techniques, Methods in Enzymology Volume 152,(Academic Press, Inc., San Diego, Calif.) (“Berger”); Sambrook et al.,Molecular Cloning—A Laboratory Manual, 2d ed., Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1989 (“Sambrook”) andCurrent Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (supplemented through 1999)(“Ausubel”). Examples of protocols sufficient to direct persons of skillthrough in vitro amplification methods, including the polymerase chainreaction (PCR), the ligase chain reaction (LCR), Qβ-replicaseamplification and other RNA polymerase mediated techniques (e.g.,NASBA), e.g., for the production of the homologous nucleic acids of thedisclosure are found in Berger, Sambrook, and Ausubel, as well as inMullis et al. (1987) U.S. Pat. No. 4,683,202; Innis et al., eds. (1990)PCR Protocols: A Guide to Methods and Applications (Academic Press Inc.San Diego, Calif.) (“Innis”); Arnheim & Levinson (Oct. 1, 1990) C&EN36-47; The Journal Of NIH Research (1991) 3: 81-94; Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli et al. (1990) Proc. Nat'l.Acad. Sci. USA 87: 1874; Lomell et al. (1989) J. Clin. Chem. 35: 1826;Landegren et al. (1988) Science 241: 1077-1080; Van Brunt (1990)Biotechnology 8: 291-294; Wu and Wallace (1989) Gene 4:560; Barringer etal. (1990) Gene 89:117; and Sooknanan and Malek (1995) Biotechnology 13:563-564. Improved methods for cloning in vitro amplified nucleic acidsare described in Wallace et al., U.S. Pat. No. 5,426,039. Improvedmethods for amplifying large nucleic acids by PCR are summarized inCheng et al. (1994) Nature 369: 684-685 and the references citedtherein, in which PCR amplicons of up to 40 kb are generated. One ofskill will appreciate that essentially any RNA can be converted into adouble stranded DNA suitable for restriction digestion, PCR expansionand sequencing using reverse transcriptase and a polymerase. See, e.g.,Ausubel, Sambrook and Berger, all supra.

The disclosure provides accession numbers for various genes, homologsand variants useful in the generation of recombinant microorganismdescribed herein. It is to be understood that homologs and variantsdescribed herein are exemplary and non-limiting. Additional homologs,variants and sequences are available to those of skill in the art usingvarious databases including, for example, the National Center forBiotechnology Information (NCBI) access to which is available on theWorld-Wide-Web.

Ethanol Dehydrogenase (also referred to as Aldehyde-alcoholdehydrogenase) is encoded in E. coli by adhE. adhE comprises threeactivities: alcohol dehydrogenase (ADH); acetaldehyde/acetyl-CoAdehydrogenase (ACDH); pyruvate-formate-lyase deactivase (PFLdeactivase); PFL deactivase activity catalyzes the quenching of thepyruvate-formate-lyase catalyst in an iron, NAD, and CoA dependentreaction. Homologs are known in the art (see, e.g., aldehyde-alcoholdehydrogenase (Polytomella sp. Pringsheim 198.80)gi|40644910|emb|CAD42653.2|(40644910); aldehyde-alcohol dehydrogenase(Clostridium botulinum A str. ATCC 3502)gi|148378348|ref|YP_(—)001252889.1|(148378348); aldehyde-alcoholdehydrogenase (Yersinia pestis CO92)gi|16122410|ref|NP_(—)405723.1|(16122410); aldehyde-alcoholdehydrogenase (Yersinia pseudotuberculosis IP 32953)gi|51596429|ref|YP_(—)070620.1|(51596429); aldehyde-alcoholdehydrogenase (Yersinia pestis CO92)gi|115347889|emb|CAL20810.1|(115347889); aldehyde-alcohol dehydrogenase(Yersinia pseudotuberculosis IP 32953)gi|51589711|emb|CAH21341.1|(51589711); Aldehyde-alcohol dehydrogenase(Escherichia coli CFT073)gi|26107972|gb|AAN80172.1|AE016760_(—)31(26107972); aldehyde-alcoholdehydrogenase (Yersinia pestis biovar Microtus str. 91001)gi|45441777|ref|NP_(—)993316.1|(45441777); aldehyde-alcoholdehydrogenase (Yersinia pestis biovar Microtus str. 91001)gi|45436639|gb|AAS62193.1|(45436639); aldehyde-alcohol dehydrogenase(Clostridium perfringens ATCC 13124)gi|110798574|ref|YP_(—)697219.1|(110798574); aldehyde-alcoholdehydrogenase (Shewanella oneidensisMR-1)gi|24373696|ref|NP_(—)717739.1|(24373696); aldehyde-alcoholdehydrogenase (Clostridium botulinum A str. ATCC 19397)gi|153932445|ref|YP_(—)001382747.1|(153932445); aldehyde-alcoholdehydrogenase (Yersinia pestis biovar Antigua str. E1979001)gi|165991833|gb|EDR44134.1|(165991833); aldehyde-alcohol dehydrogenase(Clostridium botulinum A str. Hall)gi|153937530|ref|YP_(—)001386298.1|(153937530); aldehyde-alcoholdehydrogenase (Clostridium perfringens ATCC 13124)gi|110673221|gb|ABG82208.1|(110673221); aldehyde-alcohol dehydrogenase(Clostridium botulinum A str. Hall)gi|152933444|gb|ABS38943.1|(152933444); aldehyde-alcohol dehydrogenase(Yersinia pestis biovar Orientalis str. F1991016)gi|165920640|gb|EDR37888.1|(165920640); aldehyde-alcohol dehydrogenase(Yersinia pestis biovar Orientalis str.IP275)gi|165913933|gb|EDR32551.1|(165913933); aldehyde-alcoholdehydrogenase (Yersinia pestis Angola)gi|162419116|ref|YP_(—)001606617.1|(162419116); aldehyde-alcoholdehydrogenase (Clostridium botulinum F str. Langeland)gi|153940830|ref|YP_(—)001389712.1|(153940830); aldehyde-alcoholdehydrogenase (Escherichia coli HS)gi|157160746|ref|YP_(—)001458064.1|(157160746); aldehyde-alcoholdehydrogenase (Escherichia coli E24377A)gi|157155679|ref|YP_(—)001462491.1|(157155679); aldehyde-alcoholdehydrogenase (Yersinia enterocolitica subsp. enterocolitica 8081)gi|123442494|ref|YP_(—)001006472.1|(123442494); aldehyde-alcoholdehydrogenase (Synechococcus sp. JA-3-3Ab)gi|86605191|ref|YP_(—)473954.1|(86605191); aldehyde-alcoholdehydrogenase (Listeria monocytogenes str. 4b F2365)gi|46907864|ref|YP_(—)014253.1|(46907864); aldehyde-alcoholdehydrogenase (Enterococcus faecalis V583)gi|29375484|ref|NP_(—)814638.1|(29375484); aldehyde-alcoholdehydrogenase (Streptococcus agalactiae 2603V/R)gi|22536238|ref|NP_(—)687089.1|(22536238); aldehyde-alcoholdehydrogenase (Clostridium botulinum A str. ATCC 19397)gi|152928489|gb|ABS33989.1|(152928489); aldehyde-alcohol dehydrogenase(Escherichia coli E24377A) gi|157077709|gb|ABV17417.1|(157077709);aldehyde-alcohol dehydrogenase (Escherichia coli HS)gi|157066426|gb|ABV05681.1|(157066426); aldehyde-alcohol dehydrogenase(Clostridium botulinum F str. Langeland)gi|152936726|gb|ABS42224.1|(152936726); aldehyde-alcohol dehydrogenase(Yersinia pestis CA88-4125) gi|149292312|gb|EDM42386.1|(149292312);aldehyde-alcohol dehydrogenase (Yersinia enterocolitica subsp.enterocolitica 8081) gi|122089455|emb|CAL12303.1|(122089455);aldehyde-alcohol dehydrogenase (Chlamydomonas reinhardtii)gi|92084840|emb|CAF04128.1|(92084840); aldehyde-alcohol dehydrogenase(Synechococcus sp. JA-3-3Ab) gi|86553733|gb|ABC98691.1|(86553733);aldehyde-alcohol dehydrogenase (Shewanella oneidensis MR-1)gi|24348056|gb|AAN55183.1|AE015655_(—)9(24348056); aldehyde-alcoholdehydrogenase (Enterococcus faecalis V583)gi|29342944|gb|AAO80708.1|(29342944); aldehyde-alcohol dehydrogenase(Listeria monocytogenes str. 4b F2365)gi|46881133|gb|AAT04430.1|(46881133); aldehyde-alcohol dehydrogenase(Listeria monocytogenes str. 1/2a F6854)gi|47097587|ref|ZP_(—)00235115.1|(47097587); aldehyde-alcoholdehydrogenase (Listeria monocytogenes str. 4b H7858)gi|47094265|ref|ZP_(—)00231973.1|(47094265); aldehyde-alcoholdehydrogenase (Listeria monocytogenes str. 4b H7858)gi|47017355|gb|EAL08180.1|(47017355); aldehyde-alcohol dehydrogenase(Listeria monocytogenes str. 1/2a F6854)gi|47014034|gb|EAL05039.1|(47014034); aldehyde-alcohol dehydrogenase(Streptococcus agalactiae 2603V/R)gi|22533058|gb|AAM98961.1|AE014194_(—)6(22533058)p; aldehyde-alcoholdehydrogenase (Yersinia pestis biovar Antigua str. E1979001)gi|166009278|ref|ZP_(—)02230176.1|(166009278); aldehyde-alcoholdehydrogenase (Yersinia pestis biovar Orientalis str. IP275)gi|165938272|ref|ZP_(—)02226831.1|(165938272); aldehyde-alcoholdehydrogenase (Yersinia pestis biovar Orientalis str. F1991016)gi|165927374|ref|ZP_(—)02223206.1|(165927374); aldehyde-alcoholdehydrogenase (Yersinia pestis Angola)gi|162351931|gb|ABX85879.1|(162351931); aldehyde-alcohol dehydrogenase(Yersinia pseudotuberculosis IP 31758)gi|153949366|ref|YP_(—)001400938.1|(153949366); aldehyde-alcoholdehydrogenase (Yersinia pseudotuberculosis IP 31758)gi|152960861|gb|ABS48322.1|(152960861); aldehyde-alcohol dehydrogenase(Yersinia pestis CA88-4125)gi|149365899|ref|ZP_(—)01887934.1|(149365899); Acetaldehydedehydrogenase (acetylating) (Escherichia coli CFT073)gi|26247570|ref|NP_(—)753610.1|(26247570); aldehyde-alcoholdehydrogenase (includes: alcohol dehydrogenase; acetaldehydedehydrogenase (acetylating) (EC 1.2.1.10) (acdh); pyruvate-formate-lyasedeactivase (pfl deactivase)) (Clostridium botulinum A str. ATCC 3502)gi|148287832|emb|CAL81898.1|(148287832); aldehyde-alcohol dehydrogenase(Includes: Alcohol dehydrogenase (ADH); Acetaldehyde dehydrogenase(acetylating) (ACDH); Pyruvate-formate-lyase deactivase (PFLdeactivase)) gi|71152980|sp|P0A9Q7.2|ADHE_ECOLI(71152980);aldehyde-alcohol dehydrogenase (includes: alcohol dehydrogenase andacetaldehyde dehydrogenase, and pyruvate-formate-lyase deactivase(Erwinia carotovora subsp. atroseptica SCRI1043)gi|50121254|ref|YP_(—)050421.1|(50121254); aldehyde-alcoholdehydrogenase (includes: alcohol dehydrogenase and acetaldehydedehydrogenase, and pyruvate-formate-lyase deactivase (Erwinia carotovorasubsp. atroseptica SCRI1043) gi|49611780|emb|CAG75229.1|(49611780);Aldehyde-alcohol dehydrogenase (Includes: Alcohol dehydrogenase (ADH);Acetaldehyde dehydrogenase (acetylating) (ACDH))gi|19858620|sp|P33744.3|ADHE_CLOAB(19858620); Aldehyde-alcoholdehydrogenase (Includes: Alcohol dehydrogenase (ADH); Acetaldehydedehydrogenase (acetylating) (ACDH); Pyruvate-formate-lyase deactivase(PFL deactivase)) gi|71152683|sp|P0A9Q8.2|ADHE_ECO57(71152683);aldehyde-alcohol dehydrogenase (includes: alcohol dehydrogenase;acetaldehyde dehydrogenase (acetylating); pyruvate-formate-lyasedeactivase (Clostridium difficile 630)gi|126697906|ref|YP_(—)001086803.1|(126697906); aldehyde-alcoholdehydrogenase (includes: alcohol dehydrogenase; acetaldehydedehydrogenase (acetylating); pyruvate-formate-lyase deactivase(Clostridium difficile 630) gi|115249343|emb|CAJ67156.1|(115249343);Aldehyde-alcohol dehydrogenase (includes: alcohol dehydrogenase (ADH)and acetaldehyde dehydrogenase (acetylating) (ACDH);pyruvate-formate-lyase deactivase (PFL deactivase)) (Photorhabdusluminescens subsp. laumondii TTO1)gi|37526388|ref|NP_(—)929732.1|(37526388); aldehyde-alcoholdehydrogenase 2 (includes: alcohol dehydrogenase; acetaldehydedehydrogenase) (Streptococcus pyogenes str. Manfredo)gi|134271169|emb|CAM29381.1|(134271169); Aldehyde-alcohol dehydrogenase(includes: alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase(acetylating) (ACDH); pyruvate-formate-lyase deactivase (PFLdeactivase)) (Photorhabdus luminescens subsp. laumondii TTO1)gi|36785819|emb|CAE14870.1|(36785819); aldehyde-alcohol dehydrogenase(includes: alcohol dehydrogenase and pyruvate-formate-lyase deactivase(Clostridium difficile 630)gi|126700586|ref|YP_(—)001089483.1|(126700586); aldehyde-alcoholdehydrogenase (includes: alcohol dehydrogenase andpyruvate-formate-lyase deactivase (Clostridium difficile 630)gi|115252023|emb|CAJ69859.1|(115252023); aldehyde-alcohol dehydrogenase2 (Streptococcus pyogenes str. Manfredo)gi|139472923|ref|YP_(—)001127638.1|(139472923); aldehyde-alcoholdehydrogenase E (Clostridium perfringens str. 13)gi|18311513|ref|NP_(—)563447.1|(18311513); aldehyde-alcoholdehydrogenase E (Clostridium perfringens str. 13)gi|18146197|dbj|BAB82237.1|(18146197); Aldehyde-alcohol dehydrogenase,ADHE1 (Clostridium acetobutylicum ATCC 824)gi|15004739|ref|NP_(—)149199.1|(15004739); Aldehyde-alcoholdehydrogenase, ADHE1 (Clostridium acetobutylicum ATCC 824)gi|14994351|gb|AAK76781.1|AE001438_(—)34(14994351); Aldehyde-alcoholdehydrogenase 2 (Includes: Alcohol dehydrogenase (ADH);acetaldehyde/acetyl-CoA dehydrogenase (ACDH))gi|2492737|sp|Q24803.1|ADH2_ENTHI(2492737); alcohol dehydrogenase(Salmonella enterica subsp. enterica serovar Typhi str. CT18)gi|16760134|ref|NP_(—)455751.1|(16760134); and alcohol dehydrogenase(Salmonella enterica subsp. enterica serovar Typhi)gi|16502428|emb|CAD08384.1|(16502428)), each sequence associated withthe accession number is incorporated herein by reference in itsentirety.

Lactate Dehydrogenase (also referred to as D-lactate dehydrogenase andfermentive dehydrognase) is encoded in E. coli by ldhA and catalyzes theNADH-dependent conversion of pyruvate to D-lactate. ldhA homologs andvariants are known. In fact there are currently 1664 bacterial lactatedehydrogenases available through NCBI. For example, such homologs andvariants include, for example, D-lactate dehydrogenase (D-LDH)(Fermentative lactate dehydrogenase)gi|1730102|sp|P52643.1|LDHD_ECOLI(1730102); D-lactate dehydrogenasegi|1049265|gb|AAB51772.1|(1049265); D-lactate dehydrogenase (Escherichiacoli APEC O1) gi|117623655|ref|YP_(—)852568.1|(117623655); D-lactatedehydrogenase (Escherichia coli CFT073)gi|26247689|ref|NP_(—)753729.1|(26247689); D-lactate dehydrogenase(Escherichia coli O157:H7 EDL933)gi|15801748|ref|NP_(—)287766.1|(15801748); D-lactate dehydrogenase(Escherichia coli APEC O1) gi|115512779|gb|ABJ00854.1|(115512779);D-lactate dehydrogenase (Escherichia coli CFT073)gi|26108091|gb|AAN80291.1|AE016760_(—)150(26108091); fermentativeD-lactate dehydrogenase, NAD-dependent (Escherichia coli K12)gi|16129341|ref|NP_(—)415898.1|(16129341); fermentative D-lactatedehydrogenase, NAD-dependent (Escherichia coli UTI89)gi|91210646|ref|YP_(—)540632.1|(91210646); fermentative D-lactatedehydrogenase, NAD-dependent (Escherichia coli K12)gi|1787645|gb|AAC74462.1|(1787645); fermentative D-lactatedehydrogenase, NAD-dependent (Escherichia coli W3110)gi|89108227|ref|AP_(—)002007.1|(89108227); fermentative D-lactatedehydrogenase, NAD-dependent (Escherichia coli W3110)gi|1742259|dbj|BAA14990.1|(1742259); fermentative D-lactatedehydrogenase, NAD-dependent (Escherichia coli UTI89)gi|91072220|gb|ABE07101.1|(91072220); fermentative D-lactatedehydrogenase, NAD-dependent (Escherichia coli O157:H7 EDL933)gi|12515320|gb|AAG56380.1|AE005366_(—)6(12515320); fermentativeD-lactate dehydrogenase (Escherichia coli O157:H7 str. Sakai)gi|13361468|dbj|BAB35425.1|(13361468); COG1052: Lactate dehydrogenaseand related dehydrogenases (Escherichia coli 101-1)gi|83588593|ref|ZP_(—)00927217.1|(83588593); COG1052: Lactatedehydrogenase and related dehydrogenases (Escherichia coli 53638)gi|75515985|ref|ZP_(—)00738103.1|(75515985); COG1052: Lactatedehydrogenase and related dehydrogenases (Escherichia coli E22)gi|75260157|ref|ZP_(—)00731425.1|(75260157); COG1052: Lactatedehydrogenase and related dehydrogenases (Escherichia coli F11)gi|75242656|ref|ZP_(—)00726400.1|(75242656); COG1052: Lactatedehydrogenase and related dehydrogenases (Escherichia coli E110019)gi|75237491|ref|ZP_(—)00721524.1|(75237491); COG1052: Lactatedehydrogenase and related dehydrogenases (Escherichia coli B7A)gi|75231601|ref|ZP_(—)00717959.1|(75231601); and COG1052: Lactatedehydrogenase and related dehydrogenases (Escherichia coli B171)gi|75211308|ref|ZP_(—)00711407.1|(75211308), each sequence associatedwith the accession number is incorporated herein by reference in itsentirety.

Two membrane-bound, FAD-containing enzymes are responsible for thecatalysis of fumarate and succinate interconversion; the fumaratereductase is used in anaerobic growth, and the succinate dehydrogenaseis used in aerobic growth. Fumarate reductase comprises multiplesubunits (e.g., frdA, B, and C in E. coli). Modification of any one ofthe subunits can result in the desired activity herein. For example, aknockout of frdB, frdC or frdBC is useful in the methods of thedisclosure. Frd homologs and variants are known. For example, homologsand variants includes, for example, Fumarate reductase subunit D(Fumarate reductase 13 kDa hydrophobic protein)gi|67463543|sp|P0A8Q3.1|FRDD_ECOLI(67463543); Fumarate reductase subunitC (Fumarate reductase 15 kDa hydrophobic protein)gi|1346037|sp|P20923.2|FRDC_PROVU(1346037); Fumarate reductase subunit D(Fumarate reductase 13 kDa hydrophobic protein)gi|120499|sp|P20924.1|FRDD_PROVU(120499); Fumarate reductase subunit C(Fumarate reductase 15 kDa hydrophobic protein)gi|67463538|sp|P0A8Q0.1|FRDC_ECOLI(67463538); fumarate reductaseiron-sulfur subunit (Escherichia coli) gi|145264|gb|AAA23438.1|(145264);fumarate reductase flavoprotein subunit (Escherichia coli)gi|145263|gb|AAA23437.1|(145263); Fumarate reductase flavoproteinsubunit gi|37538290|sp|P17412.3|FRDA_WOLSU(37538290); Fumarate reductaseflavoprotein subunit gi|120489|sp|P00363.3|FRDA_ECOLI(120489); Fumaratereductase flavoprotein subunit gi|120490|sp|P20922.1|FRDA_PROVU(120490);Fumarate reductase flavoprotein subunit precursor (Flavocytochrome c)(Flavocytochrome c3) (Fcc3)gi|119370087|sp|Q07WU7.2|FRDA_SHEFN(119370087); Fumarate reductaseiron-sulfur subunit gi|81175308|sp|P0AC47.2|FRDB_ECOLI(81175308);Fumarate reductase flavoprotein subunit (Flavocytochrome c)(Flavocytochrome c3) (Fcc3)gi|119370088|sp|P0C278.1|FRDA_SHEFR(119370088); Frd operonuncharacterized protein C gi|140663|sp|P20927.1|YFRC_PROVU(140663); Frdoperon probable iron-sulfur subunit Agi|140661|sp|P20925.1|YFRA_PROVU(140661); Fumarate reductase iron-sulfursubunit gi|120493|sp|P20921.2|FRDB_PROVU(120493); Fumarate reductaseflavoprotein subunit gi|2494617|sp|O06913.2|FRDA_HELPY(2494617);Fumarate reductase flavoprotein subunit precursor (Iron(III)-inducedflavocytochrome C3) (Ifc3) gi|13878499|sp|Q9Z4P0.1|FRD2_SHEFN(13878499);Fumarate reductase flavoprotein subunitgi|54041009|sp|P64174.1|FRDA_MYCTU(54041009); Fumarate reductaseflavoprotein subunit gi|54037132|sp|P64175.1|FRDA_MYCBO(54037132);Fumarate reductase flavoprotein subunitgi|12230114|sp|Q9ZMP0.1|FRDA_HELPJ(12230114); Fumarate reductaseflavoprotein subunit gi|1169737|sp|P44894.1|FRDA_HAEIN(1169737);fumarate reductase flavoprotein subunit (Wolinella succinogenes)gi|13160058|emb|CAA04214.2|(13160058); Fumarate reductase flavoproteinsubunit precursor (Flavocytochrome c) (FL cyt)gi|25452947|sp|P83223.2|FRDA_SHEON(25452947); fumarate reductaseiron-sulfur subunit (Wolinella succinogenes)gi|2282000|emb|CAA04215.1|(2282000); and fumarate reductase cytochrome bsubunit (Wolinella succinogenes) gi|2281998|emb|CAA04213.1|(2281998),each sequence associated with the accession number is incorporatedherein by reference in its entirety.

Acetate kinase is encoded in E. coli by ackA. AckA is involved inconversion of acetyl-coA to acetate. Specifically, ackA catalyzes theconversion of acetyl-phophate to acetate. AckA homologs and variants areknown. The NCBI database list approximately 1450 polypeptides asbacterial acetate kinases. For example, such homologs and variantsinclude acetate kinase (Streptomyces coelicolor A3(2))gi|21223784|ref|NP_(—)629563.1|(21223784); acetate kinase (Streptomycescoelicolor A3(2)) gi|6808417|emb|CAB70654.1|(6808417); acetate kinase(Streptococcus pyogenes M1 GAS)gi|15674332|ref|NP_(—)268506.1|(15674332); acetate kinase (Campylobacterjejuni subsp. jejuni NCTC 11168)gi|15792038|ref|NP_(—)281861.1|(15792038); acetate kinase (Streptococcuspyogenes M1 GAS) gi|13621416|gb|AAK33227.1|(13621416); acetate kinase(Rhodopirellula baltica SH 1) gi|32476009|ref|NP_(—)869003.1|(32476009);acetate kinase (Rhodopirellula baltica SH 1)gi|32472045|ref|NP_(—)865039.1|(32472045); acetate kinase (Campylobacterjejuni subsp. jejuni NCTC 11168)gi|112360034|emb|CAL34826.1|(112360034); acetate kinase (Rhodopirellulabaltica SH 1) gi|32446553|emb|CAD76388.1|(32446553); acetate kinase(Rhodopirellula baltica SH 1) gi|32397417|emb|CAD72723.1|(32397417);AckA (Clostridium kluyveri DSM 555)gi|153954016|ref|YP_(—)001394781.1|(153954016); acetate kinase(Bifidobacterium longum NCC2705)gi|23465540|ref|NP_(—)696143.1|(23465540); AckA (Clostridium kluyveriDSM 555) gi|46346897|gb|EDK33433.1|(146346897); Acetate kinase(Corynebacterium diphtheriae) gi|38200875|emb|CAE50580.1|(38200875);acetate kinase (Bifidobacterium longum NCC2705)gi|23326203|gb|AAN24779.1|(23326203); Acetate kinase (Acetokinase)gi|67462089|sp|P0A6A3.1|ACKA_ECOLI(67462089); and AckA (Bacilluslicheniformis DSM 13) gi|52349315|gb|AAU41949.1|(52349315), thesequences associated with such accession numbers are incorporated hereinby reference.

Phosphate acetyltransferase is encoded in E. coli by pta. PTA isinvolved in conversion of acetate to acetyl-CoA. Specifically, PTAcatalyzes the conversion of acetyl-coA to acetyl-phosphate. PTA homologsand variants are known. There are approximately 1075 bacterial phosphateacetyltransferases available on NCBI. For example, such homologs andvariants include phosphate acetyltransferase Pta (Rickettsia felisURRWXCal2) gi|67004021|gb|AAY60947.1|(67004021); phosphateacetyltransferase (Buchnera aphidicola str. Cc (Cinara cedri))gi|116256910|gb|ABJ90592.1|(116256910); pta (Buchnera aphidicola str. Cc(Cinara cedri)) gi|116515056|ref|YP_(—)802685.1|(116515056); pta(Wigglesworthia glossinidia endosymbiont of Glossina brevipalpis)gi|25166135|dbj|BAC24326.1|(25166135); Pta (Pasteurella multocida subsp.multocida str. Pm70) gi|12720993|gb|AAK02789.1|(12720993); Pta(Rhodospirillum rubrum) gi|25989720|gb|AAN75024.1|(25989720); pta(Listeria welshimeri serovar 6b str. SLCC5334)gi|116742418|emb|CAK21542.1|(116742418); Pta (Mycobacterium avium subsp.paratuberculosis K-10) gi|41398816|gb|AAS06435.1|(41398816); phosphateacetyltransferase (pta) (Borrelia burgdorferi B31)gi|15594934|ref|NP_(—)212723.1|(15594934); phosphate acetyltransferase(pta) (Borrelia burgdorferi B31) gi|2688508|gb|AAB91518.1|(2688508);phosphate acetyltransferase (pta) (Haemophilus influenzae Rd KW20)gi|1574131|gb|AAC22857.1|(1574131); Phosphate acetyltransferase Pta(Rickettsia bellii RML369-C) gi|91206026|ref|YP_(—)538381.1|(91206026);Phosphate acetyltransferase Pta (Rickettsia bellii RML369-C)gi|91206025|ref|YP_(—)538380.1|(91206025); phosphate acetyltransferasepta (Mycobacterium tuberculosis F11)gi|148720131|gb|ABR04756.1|(148720131); phosphate acetyltransferase pta(Mycobacterium tuberculosis str. Haarlem)gi|134148886|gb|EBA40931.1|(134148886); phosphate acetyltransferase pta(Mycobacterium tuberculosis C) gi|124599819|gb|EAY58829.1|(124599819);Phosphate acetyltransferase Pta (Rickettsia bellii RML369-C)gi|91069570|gb|ABE05292.1|(91069570); Phosphate acetyltransferase Pta(Rickettsia bellii RML369-C) gi|91069569|gb|ABE05291.1|(91069569);phosphate acetyltransferase (pta) (Treponema pallidum subsp. pallidumstr. Nichols) gi|5639088|ref|NP_(—)218534.1|(15639088); and phosphateacetyltransferase (pta) (Treponema pallidum subsp. pallidum str.Nichols) gi|3322356|gb|AAC65090.1|(3322356), each sequence associatedwith the accession number is incorporated herein by reference in itsentirety.

Pyruvate-formate lyase (Formate acetylytransferase) is an enzyme thatcatalyzes the conversion of pyruvate to acetly-coA and formate. It isinduced by pfl-activating enzyme under anaerobic conditions bygeneration of an organic free radical and decreases significantly duringphosphate limitation. Formate acetylytransferase is encoded in E. coliby pflB. PFLB homologs and variants are known. For examples, suchhomologs and variants include, for example, Formate acetyltransferase 1(Pyruvate formate-lyase 1) gi|129879|sp|P09373.2|PFLB_ECOLI(129879);formate acetyltransferase 1 (Yersinia pestis CO92)gi|16121663|ref|NP_(—)404976.1|(16121663); formate acetyltransferase 1(Yersinia pseudotuberculosis IP 32953)gi|51595748|ref|YP_(—)069939.1|(51595748); formate acetyltransferase 1(Yersinia pestis biovar Microtus str. 91001)gi|45441037|ref|NP_(—)992576.1|(45441037); formate acetyltransferase 1(Yersinia pestis CO92) gi|115347142|emb|CAL20035.1|(115347142); formateacetyltransferase 1 (Yersinia pestis biovar Microtus str. 91001)gi|45435896|gb|AAS61453.1|(45435896); formate acetyltransferase 1(Yersinia pseudotuberculosis IP 32953)gi|51589030|emb|CAH20648.1|(51589030); formate acetyltransferase 1(Salmonella enterica subsp. enterica serovar Typhi str. CT18)gi|16759843|ref|NP_(—)455460.1|(16759843); formate acetyltransferase 1(Salmonella enterica subsp. enterica serovar Paratyphi A str. ATCC 9150)gi|56413977|ref|YP_(—)151052.1|(56413977); formate acetyltransferase 1(Salmonella enterica subsp. enterica serovar Typhi)gi|16502136|emb|CAD05373.1|(16502136); formate acetyltransferase 1(Salmonella enterica subsp. enterica serovar Paratyphi A str. ATCC 9150)gi|56128234|gb|AAV77740.1|(56128234); formate acetyltransferase 1(Shigella dysenteriae Sd197) gi|82777577|ref|YP_(—)403926.1|(82777577);formate acetyltransferase 1 (Shigella flexneri 2a str. 2457T)gi|30062438|ref|NP_(—)836609.1|(30062438); formate acetyltransferase 1(Shigella flexneri 2a str. 2457T) gi|30040684|gb|AAP16415.1|(30040684);formate acetyltransferase 1 (Shigella flexneri 5 str. 8401)gi|110614459|gb|ABF03126.1|(110614459); formate acetyltransferase 1(Shigella dysenteriae Sd197) gi|81241725|gb|ABB62435.1|(81241725);formate acetyltransferase 1 (Escherichia coli O157:H7 EDL933)gi|12514066|gb|AAG55388.1|AE005279_(—)8(12514066); formateacetyltransferase 1 (Yersinia pestis KIM)gi|22126668|ref|NP_(—)670091.1|(22126668); formate acetyltransferase 1(Streptococcus agalactiae A909)gi|76787667|ref|YP_(—)330335.1|(76787667); formate acetyltransferase 1(Yersinia pestis KIM) gi|21959683|gb|AAM86342.1|AE013882_(—)3(21959683);formate acetyltransferase 1 (Streptococcus agalactiae A909)gi|76562724|gb|ABA45308.1|(76562724); formate acetyltransferase 1(Yersinia enterocolitica subsp. enterocolitica 8081)gi|123441844|ref|YP_(—)001005827.1|(123441844); formateacetyltransferase 1 (Shigella flexneri 5 str. 8401)gi|110804911|ref|YP_(—)688431.1|(110804911); formate acetyltransferase 1(Escherichia coli UTI89) gi|91210004|ref|YP_(—)539990.1|(91210004);formate acetyltransferase 1 (Shigella boydii Sb227)gi|82544641|ref|YP_(—)408588.1|(82544641); formate acetyltransferase 1(Shigella sonnei Ss046) gi|74311459|ref|YP_(—)309878.1|(74311459);formate acetyltransferase 1 (Klebsiella pneumoniae subsp. pneumoniae MGH78578) gi|152969488|ref|YP_(—)001334597.1|(152969488); formateacetyltransferase 1 (Salmonella enterica subsp. enterica serovar TyphiTy2) gi|29142384|ref|NP_(—)805726.1|(29142384) formate acetyltransferase1 (Shigella flexneri 2a str. 301)gi|24112311|ref|NP_(—)706821.1|(24112311); formate acetyltransferase 1(Escherichia coli O157:H7 EDL933)gi|15800764|ref|NP_(—)286778.1|(15800764); formate acetyltransferase 1(Klebsiella pneumoniae subsp. pneumoniae MGH 78578)gi|150954337|gb|ABR76367.1|(150954337); formate acetyltransferase 1(Yersinia pestis CA88-4125)gi|149366640|ref|ZP_(—)01888674.1|(149366640); formate acetyltransferase1 (Yersinia pestis CA88-4125) gi|149291014|gb|EDM41089.1|(149291014);formate acetyltransferase 1 (Yersinia enterocolitica subsp.enterocolitica 8081) gi|122088805|emb|CAL11611.1|(122088805); formateacetyltransferase 1 (Shigella sonnei Ss046)gi|73854936|gb|AAZ87643.1|(73854936); formate acetyltransferase 1(Escherichia coli UTI89) gi|91071578|gb|ABE06459.1|(91071578); formateacetyltransferase 1 (Salmonella enterica subsp. enterica serovar TyphiTy2) gi|29138014|gb|AAO69575.1|(29138014); formate acetyltransferase 1(Shigella boydii Sb227) gi|81246052|gb|ABB66760.1|(81246052); formateacetyltransferase 1 (Shigella flexneri 2a str. 301)gi|24051169|gb|AAN42528.1|(24051169); formate acetyltransferase 1(Escherichia coli O157:H7 str. Sakai)gi|13360445|dbj|BAB34409.1|(13360445); formate acetyltransferase 1(Escherichia coli O157:H7 str. Sakai)gi|15830240|ref|NP_(—)309013.1|(15830240); formate acetyltransferase I(pyruvate formate-lyase 1) (Photorhabdus luminescens subsp. laumondiiTTO1) gi|36784986|emb|CAE13906.1|(36784986); formate acetyltransferase I(pyruvate formate-lyase 1) (Photorhabdus luminescens subsp. laumondiiTTO1) gi|37525558|ref|NP_(—)928902.1|(37525558); formateacetyltransferase (Staphylococcus aureus subsp. aureus Mu50)gi|14245993|dbj|BAB56388.1|(14245993); formate acetyltransferase(Staphylococcus aureus subsp. aureus Mu50)gi|15923216|ref|NP_(—)370750.1|(15923216); Formate acetyltransferase(Pyruvate formate-lyase) gi|81706366|sp|Q7A7X6.1|PFLB_STAAN(81706366);Formate acetyltransferase (Pyruvate formate-lyase)gi|81782287|sp|Q99WZ7.1|PFLB_STAAM(81782287); Formate acetyltransferase(Pyruvate formate-lyase) gi|81704726|sp|Q7A1W9.1|PFLB_STAAW(81704726);formate acetyltransferase (Staphylococcus aureus subsp. aureus Mu3)gi|156720691|dbj|BAF77108.1|(156720691); formate acetyltransferase(Erwinia carotovora subsp. atroseptica SCRI1043)gi|50121521|ref|YP_(—)050688.1|(50121521); formate acetyltransferase(Erwinia carotovora subsp. atroseptica SCRI1043)gi|49612047|emb|CAG75496.1|(49612047); formate acetyltransferase(Staphylococcus aureus subsp. aureus str. Newman)gi|150373174|dbj|BAF66434.1|(150373174); formate acetyltransferase(Shewanella oneidensis MR-1) gi|24374439|ref|NP_(—)718482.1|(24374439);formate acetyltransferase (Shewanella oneidensis MR-1)gi|24349015|gb|AAN55926.1|AE015730_(—)3(24349015); formateacetyltransferase (Actinobacillus pleuropneumoniae serovar 3 str. JL03)gi|165976461|ref|YP_(—)001652054.1|(165976461); formateacetyltransferase (Actinobacillus pleuropneumoniae serovar 3 str. JL03)gi|165876562|gb|ABY69610.1|(165876562); formate acetyltransferase(Staphylococcus aureus subsp. aureus MW2)gi|21203365|dbj|BAB94066.1|(21203365); formate acetyltransferase(Staphylococcus aureus subsp. aureus N315)gi|13700141|dbj|BAB41440.1|(13700141); formate acetyltransferase(Staphylococcus aureus subsp. aureus str. Newman)gi|151220374|ref|YP_(—)001331197.1|(151220374); formateacetyltransferase (Staphylococcus aureus subsp. aureus Mu3)gi|156978556|ref|YP_(—)001440815.1|(156978556); formateacetyltransferase (Synechococcus sp. JA-2-3B′ a(2-13))gi|86607744|ref|YP_(—)476506.1|(86607744); formate acetyltransferase(Synechococcus sp. JA-3-3Ab) gi|86605195|ref|YP_(—)473958.1|(86605195);formate acetyltransferase (Streptococcus pneumoniae D39)gi|116517188|ref|YP_(—)815928.1|(116517188); formate acetyltransferase(Synechococcus sp. JA-2-3B′ a(2-13))gi|86556286|gb|ABD01243.1|(86556286); formate acetyltransferase(Synechococcus sp. JA-3-3Ab) gi|86553737|gb|ABC98695.1|(86553737);formate acetyltransferase (Clostridium novyi NT)gi|118134908|gb|ABK61952.1|(118134908); formate acetyltransferase(Staphylococcus aureus subsp. aureus MRSA252)gi|49482458|ref|YP_(—)039682.1|(49482458); and formate acetyltransferase(Staphylococcus aureus subsp. aureus MRSA252)gi|49240587|emb|CAG39244.1|(49240587), each sequence associated with theaccession number is incorporated herein by reference in its entirety.

Alpha isopropylmalate synthase (EC 2.3.3.13, sometimes referred to a2-isopropylmalate synthase, alpha-IPM synthetase) catalyzes thecondensation of the acetyl group of acetyl-CoA with3-methyl-2-oxobutanoate (2-oxoisovalerate) to form3-carboxy-3-hydroxy-4-methylpentanoate (2-isopropylmalate). Alphaisopropylmalate synthase is encoded in E. coli by leuA. LeuA homologsand variants are known. For example, such homologs and variants include,for example, 2-isopropylmalate synthase (Corynebacterium glutamicum)gi|452382|emb|CAA50295.1|(452382); 2-isopropylmalate synthase(Escherichia coli K12) gi|16128068|ref|NP_(—)414616.1|(16128068);2-isopropylmalate synthase (Escherichia coli K12)gi|1786261|gb|AAC73185.1|(1786261); 2-isopropylmalate synthase(Arabidopsis thaliana) gi|15237194|ref|NP_(—)197692.1|(15237194);2-isopropylmalate synthase (Arabidopsis thaliana)gi|42562149|ref|NP_(—)173285.2|(42562149); 2-isopropylmalate synthase(Arabidopsis thaliana) gi|15221125|ref|NP_(—)177544.1|(15221125);2-isopropylmalate synthase (Streptomyces coelicolor A3(2))gi|32141173|ref|NP_(—)733575.1|(32141173); 2-isopropylmalate synthase(Rhodopirellula baltica SH 1) gi|32477692|ref|NP_(—)870686.1|(32477692);2-isopropylmalate synthase (Rhodopirellula baltica SH 1)gi|32448246|emb|CAD77763.1|(32448246); 2-isopropylmalate synthase(Akkermansia muciniphila ATCC BAA-835)gi|166241432|gb|EDR53404.1|(166241432); 2-isopropylmalate synthase(Herpetosiphon aurantiacus ATCC 23779)gi|159900959|ref|YP_(—)001547206.1|(159900959); 2-isopropylmalatesynthase (Dinoroseobacter shibae DFL 12)gi|159043149|ref|YP_(—)001531943.1|(159043149); 2-isopropylmalatesynthase (Salinispora arenicola CNS-205)gi|159035933|ref|YP_(—)001535186.1|(159035933); 2-isopropylmalatesynthase (Clavibacter michiganensis subsp. michiganensis NCPPB 382)gi|148272757|ref|YP_(—)001222318.1|(148272757); 2-isopropylmalatesynthase (Escherichia coli B)gi|124530643|ref|ZP_(—)01701227.1|(124530643); 2-isopropylmalatesynthase (Escherichia coli C str. ATCC 8739)gi|124499067|gb|EAY46563.1|(124499067); 2-isopropylmalate synthase(Bordetella pertussis Tohama I)gi|33591386|ref|NP_(—)879030.1|(33591386); 2-isopropylmalate synthase(Polynucleobacter necessarius STIR1)gi|164564063|ref|ZP_(—)02209880.1|(164564063); 2-isopropylmalatesynthase (Polynucleobacter necessarius STIR1)gi|164506789|gb|EDQ94990.1|(164506789); and 2-isopropylmalate synthase(Bacillus weihenstephanensis KBAB4)gi|163939313|ref|YP_(—)001644197.1|(163939313), any sequence associatedwith the accession number is incorporated herein by reference in itsentirety.

BCAA aminotransferases catalyze the formation of branched chain aminoacids (BCAA). A number of such aminotranferases are known and areexemplified by ilvE in E. coli. Exemplary homologs and variants includesequences designated by the following accession numbers: ilvE(Microcystis aeruginosa PCC 7806)gi|159026756|emb|CAO86637.1|(159026756); IlvE (Escherichia coli)gi|87117962|gb|ABD20288.1|(87117962); IlvE (Escherichia coli)gi|87117960|gb|ABD20287.1|(87117960); IlvE (Escherichia coli)gi|87117958|gb|ABD20286.1|(87117958); IlvE (Shigella flexneri)gi|87117956|gb|ABD20285.1|(87117956); IlvE (Shigella flexneri)gi|87117954|gb|ABD20284.1|(87117954); IlvE (Shigella flexneri)gi|87117952|gb|ABD20283.1|(87117952); IlvE (Shigella flexneri)gi|87117950|gb|ABD20282.1|(87117950); IlvE (Shigella flexneri)gi|87117948|gb|ABD20281.1|(87117948); IlvE (Shigella flexneri)gi|87117946|gb|ABD20280.1|(87117946); IlvE (Shigella flexneri)gi|87117944|gb|ABD20279.1|(87117944); IlvE (Shigella flexneri)gi|87117942|gb|ABD20278.1|(87117942); IlvE (Shigella flexneri)gi|87117940|gb|ABD20277.1|(87117940); IlvE (Shigella flexneri)gi|87117938|gb|ABD20276.1|(87117938); IlvE (Shigella dysenteriae)gi|87117936|gb|ABD20275.1|(87117936); IlvE (Shigella dysenteriae)gi|87117934|gb|ABD20274.1|(87117934); IlvE (Shigella dysenteriae)gi|87117932|gb|ABD20273.1|(87117932); IlvE (Shigella dysenteriae)gi|87117930|gb|ABD20272.1|(87117930); and IlvE (Shigella dysenteriae)gi|87117928|gb|ABD20271.1|(87117928), each sequence associated with theaccession number is incorporated herein by reference.

Tyrosine aminotransferases catalyzes transamination for bothdicarboxylic and aromatic amino-acid substrates. A tyrosineaminotransferase of E. coli is encoded by the gene tyrB. TyrB homologsand variants are known. For example, such homologs and variants includetyrB (Bordetella petrii) gi|163857093|ref|YP_(—)001631391.1|(163857093);tyrB (Bordetella petrii) gi|163260821|emb|CAP43123.1|(163260821);aminotransferase gi|551844|gb|AAA24704.1|(551844); aminotransferase(Bradyrhizobium sp. BTAi1) gi|146404387|gb|ABQ32893.1|(146404387);tyrosine aminotransferase TyrB (Salmonella enterica)gi|4775574|emb|CAB40973.2|(4775574); tyrosine aminotransferase(Salmonella typhimurium LT2) gi|16422806|gb|AAL23072.1|(16422806); andtyrosine aminotransferase gi|148085|gb|AAA24703.1|(148085), eachsequence of which is incorporated herein by reference.

Pyruvate oxidase catalyzes the conversion of pyruvate to acetate andCO₂. In E. coli, pyruvate oxidase is encoded by poxB. PoxB and homologsand variants thereof include, for example, pyruvate oxidase; PoxB(Escherichia coli)gi|685128|gb|AAB31180.1∥bbm|348451|bbs|154716(685128); PoxB (Pseudomonasfluorescens) gi|32815820|gb|AAP88293.1|(32815820); poxB (Escherichiacoli) gi|25269169|emb|CAD57486.1|(25269169); pyruvate dehydrogenase(Salmonella enterica subsp. enterica serovar Typhi)gi|16502101|emb|CAD05337.1|(16502101); pyruvate oxidase (Lactobacillusplantarum) gi|41691702|gb|AAS10156.1|(41691702); pyruvate dehydrogenase(Bradyrhizobium Japonicum) gi|20257167|gb|AAM12352.1|(20257167);pyruvate dehydrogenase (Yersinia pestis KIM)gi|22126698|ref|NP_(—)670121.1|(22126698); pyruvate dehydrogenase(cytochrome) (Yersinia pestis biovar Antigua str. B42003004)gi|166211240|ref|ZP_(—)02237275.1|(166211240); pyruvate dehydrogenase(cytochrome) (Yersinia pestis biovar Antigua str. B42003004)gi|166207011|gb|EDR51491.1|(166207011); pyruvate dehydrogenase(Pseudomonas syringae pv. tomato str. DC3000) gi|28869703|ref|NP792322.1|(28869703); pyruvate dehydrogenase (Salmonella typhimurium LT2)gi|16764297|ref|NP_(—)459912.1|(16764297); pyruvate dehydrogenase(Salmonella enterica subsp. enterica serovar Typhi str. CT18)gi|16759808|ref|NP_(—)455425.1|(16759808); pyruvate dehydrogenase(cytochrome) (Coxiella burnetii Dugway 5J108-111)gi|154706110|ref|YP_(—)001424132.1|(154706110); pyruvate dehydrogenase(Clavibacter michiganensis subsp. michiganensis NCPPB 382)gi|148273312|ref|YP_(—)001222873.1|(148273312); pyruvate oxidase(Lactobacillus acidophilus NCFM)gi|58338213|ref|YP_(—)194798.1|(58338213); and pyruvate dehydrogenase(Yersinia pestis CO92) gi|16121638|ref|NP_(—)404951.1|(16121638), thesequences of each accession number are incorporated herein by reference.

L-threonine 3-dehydrogenase (EC 1.1.1.103) catalyzes the conversion ofL-threonine to L-2-amino-3-oxobutanoate. The gene tdh encodes anL-threonine 3-dehydrogenase. There are approximately 700 L-threonine3-dehydrogenases from bacterial organism recognized in NCBI. Varioushomologs and variants of tdh include, for example, L-threonine3-dehydrogenase gi|135560|sp|P07913.1|TDH_ECOLI(135560); L-threonine3-dehydrogenase gi|166227854|sp|A4TSC6.1|TDH_YERPP(166227854);L-threonine 3-dehydrogenasegi|166227853|sp|A1JHX8.1|TDH_YERE8(166227853); L-threonine3-dehydrogenase gi|166227852|sp|A6UBM6.1|TDH_SINMW(166227852);L-threonine 3-dehydrogenasegi|166227851|sp|A1RE07.1|TDH_SHESW(166227851); L-threonine3-dehydrogenase gi|166227850|sp|A0L2Q3.1|TDH_SHESA(166227850);L-threonine 3-dehydrogenasegi|166227849|sp|A4YCC5.1|TDH_SHEPC(166227849); L-threonine3-dehydrogenase gi|166227848|sp|A3QJC8.1|TDH_SHELP(166227848);L-threonine 3-dehydrogenasegi|166227847|sp|A6WUG6.1|TDH_SHEB8(166227847); L-threonine3-dehydrogenase gi|166227846|sp|A3CYN0.1|TDH_SHEB5(166227846);L-threonine 3-dehydrogenasegi|166227845|sp|A1S1Q3.1|TDH_SHEAM(166227845); L-threonine3-dehydrogenase gi|166227844|sp|A4FND4.1|TDH_SACEN(166227844);L-threonine 3-dehydrogenasegi|166227843|sp|A1SVW5.1|TDH_PSYIN(166227843); L-threonine3-dehydrogenase gi|166227842|sp|A51GK7.1|TDH_LEGPC(166227842);L-threonine 3-dehydrogenasegi|166227841|sp|A6TFL2.1|TDH_KLEP7(166227841); L-threonine3-dehydrogenase gi|166227840|sp|A4IZ92.1|TDH_FRATW(166227840);L-threonine 3-dehydrogenasegi|166227839|sp|A0Q5K3.1|TDH_FRATN(166227839); L-threonine3-dehydrogenase gi|166227838|sp|A7NDM9.1|TDH_FRATF(166227838);L-threonine 3-dehydrogenasegi|166227837|sp|A7MID0.1|TDH_ENTS8(166227837); and L-threonine3-dehydrogenase gi|166227836|sp|A1AHF3.1|TDH_ECOK1(166227836), thesequences associated with each accession number are incorporated hereinby reference.

Acetohydroxy acid synthases (e.g. ilvH) and acetolactate synthases(e.g., alsS, ilvB, ilyl) catalyze the synthesis of the branched-chainamino acids (valine, leucine, and isoleucine). IlvH encodes anacetohydroxy acid synthase in E. coli (see, e.g., acetohydroxy acidsynthase AHAS III (IlvH) (Escherichia coli)gi|40846|emb|CAA38855.1|(40846), incorporated herein by reference).Homologs and variants as well as operons comprising ilvH are known andinclude, for example, ilvH (Microcystis aeruginosa PCC7806)gi|159026908|emb|CAO89159.1|(159026908); IlvH (Bacillusamyloliquefaciens FZB42) gi|154686966|ref|YP_(—)001422127.1|(154686966);IlvH (Bacillus amyloliquefaciens FZB42)gi|154352817|gb|ABS74896.1|(154352817); IlvH (Xenorhabdus nematophila)gi|131054140|gb|AB032787.1|(131054140); IlvH (Salmonella typhimurium)gi|7631124|gb|AAF65177.1|AF117227_(—)2(7631124), ilvN (Listeria innocua)gi|16414606|emb|CAC97322.1|(16414606); ilvN (Listeria monocytogenes)gi|16411438|emb|CAD00063.1|(16411438); acetohydroxy acid synthase(Caulobacter crescentus) gi|408939|gb|AAA23048.1|(408939); acetohydroxyacid synthase I, small subunit (Salmonella enterica subsp. entericaserovar Typhi) gi|16504830|emb|CAD03199.1|(16504830); acetohydroxy acidsynthase, small subunit (Tropheryma whipplei TW08/27)gi|28572714|ref|NP_(—)789494.1|(28572714); acetohydroxy acid synthase,small subunit (Tropheryma whipplei TW08/27)gi|28410846|emb|CAD67232.1|(28410846); acetohydroxy acid synthase I,small subunit (Salmonella enterica subsp. enterica serovar Paratyphi Astr. ATCC 9150) gi|56129933|gb|AAV79439.1|(56129933); acetohydroxy acidsynthase small subunit; acetohydroxy acid synthase, small subunitgi|551779|gb|AAA62430.1|(551779); acetohydroxy acid synthase I, smallsubunit (Salmonella enterica subsp. enterica serovar Typhi Ty2)gi|29139650|gb|AAO71216.1|(29139650); acetohydroxy acid synthase smallsubunit (Streptomyces cinnamonensis)gi|5733116|gb|AAD49432.1|AF175526_(—)1(5733116); acetohydroxy acidsynthase large subunit; and acetohydroxy acid synthase, large subunitgi|400334|gb|AAA62429.1|(400334), the sequences associated with theaccession numbers are incorporated herein by reference. Acetolactatesynthase genes include alsS and ilvI. Homologs of ilvI and alsS areknown and include, for example, acetolactate synthase small subunit(Bifidobacterium longum NCC2705) gi|23325489|gb|AAN24137.1|(23325489);acetolactate synthase small subunit (Geobacillus stearothermophilus)gi|19918933|gb|AAL99357.1|(19918933); acetolactate synthase (Azoarcussp. BH72) gi|119671178|emb|CAL95091.1|(119671178); Acetolactate synthasesmall subunit (Corynebacterium diphtheriae)gi|38199954|emb|CAE49622.1|(38199954); acetolactate synthase (Azoarcussp. BH72) gi|119669739|emb|CAL93652.1|(119669739); acetolactate synthasesmall subunit (Corynebacterium jeikeium K411)gi|68263981|emb|CAI37469.1|(68263981); acetolactate synthase smallsubunit (Bacillus subtilis) gi|1770067|emb|CAA99562.1|(1770067);Acetolactate synthase isozyme 1 small subunit (AHAS-I)(Acetohydroxy-acid synthase I small subunit) (ALS-I)gi|83309006|sp|P0ADF8.1|ILVN_ECOLI(83309006); acetolactate synthaselarge subunit (Geobacillus stearothermophilus)gi|19918932|gb|AAL99356.1|(19918932); and Acetolactate synthase, smallsubunit (Thermoanaerobacter tengcongensis MB4)gi|20806556|ref|NP_(—)621727.1|(20806556), the sequences associated withthe accession numbers are incorporated herein by reference. There areapproximately 1120 ilvB homologs and variants listed in NCBI.

Acetohydroxy acid isomeroreductase is the second enzyme in parallelpathways for the biosynthesis of isoleucine and valine. IlvC encodes anacetohydroxy acid isomeroreductase in E. coli. Homologs and variants ofilvC are known and include, for example, acetohydroxyacidreductoisomerase (Schizosaccharomyces pombe 972h-)gi|162312317|ref|NP_(—)001018845.2|(162312317); acetohydroxyacidreductoisomerase (Schizosaccharomyces pombe)gi|3116142|emb|CAA18891.1|(3116142); acetohydroxyacid reductoisomerase(Saccharomyces cerevisiae YJM789)gi|151940879|gb|EDN59261.1|(151940879); Ilv5p: acetohydroxyacidreductoisomerase (Saccharomyces cerevisiae)gi|609403|gb|AAB67753.1|(609403); ACL198Wp (Ashbya gossypii ATCC 10895)gi|45185490|ref|NP_(—)983206.1|(45185490); ACL198Wp (Ashbya gossypiiATCC 10895) gi|44981208|gb|AAS51030.1|(44981208); acetohydroxy-acidisomeroreductase; Ilv5x (Saccharomyces cerevisiae)gi|957238|gb|AAB33579.1∥bbm|369068|bbs|165406(957238); acetohydroxy-acidisomeroreductase; Ilv5g (Saccharomyces cerevisiae)gi|957236|gb|AAB33578.1∥bbm|369064|bbs|165405(957236); and ketol-acidreductoisomerase (Schizosaccharomyces pombe)gi|2696654|dbj|BAA24000.1|(2696654), each sequence associated with theaccession number is incorporated herein by reference.

Dihydroxy-acid dehydratases catalyzes the fourth step in thebiosynthesis of isoleucine and valine, the dehydratation of2,3-dihydroxy-isovaleic acid into alpha-ketoisovaleric acid. IlvD andilv3 encode a dihydroxy-acid dehydratase. Homologs and variants ofdihydroxy-acid dehydratases are known and include, for example, IlvD(Mycobacterium leprae) gi|2104594|emb|CAB08798.1|(2104594);dihydroxy-acid dehydratase (Tropheryma whipplei TW08/27)gi|28410848|emb|CAD67234.1|(28410848); dihydroxy-acid dehydratase(Mycobacterium leprae) gi|13093837|emb|CAC32140.1|(13093837);dihydroxy-acid dehydratase (Rhodopirellula baltica SH 1)gi|32447871|emb|CAD77389.1|(32447871); and putative dihydroxy-aciddehydratase (Staphylococcus aureus subsp. aureus MRSA252)gi|49242408|emb|CAG41121.1|(49242408), each sequence associated with theaccession numbers are incorporated herein by reference.

2-ketoacid decarboxylases catalyze the conversion of a 2-ketoacid to therespective aldehyde. For example, 2-ketoisovalerate decarboxylasecatalyzes the conversion of 2-ketoisovalerate to isobutyraldehyde. Anumber of 2-ketoacid decarboxylases are known and are exemplified by thepdc, pdcl, pdc5, pdc6, aro10, thI3, kdcA and kind genes. Exemplaryhomologs and variants useful for the conversion of a 2-ketoacid to therespective aldehyde comprise sequences designated by the followingaccession numbers and identified enzymatic activity:gi|44921617|gb|AAS49166.1|branched-chain alpha-keto acid decarboxylase(Lactococcus lactis); gi|15004729|ref|NP_(—)149189.1|Pyruvatedecarboxylase (Clostridium acetobutylicum ATCC 824);gi|82749898|ref|YP_(—)415639.1|probable pyruvate decarboxylase(Staphylococcus aureus RF122); gi|77961217|ref|ZP_(—)00825060.1|COG3961:Pyruvate decarboxylase and related thiamine pyrophosphate-requiringenzymes (Yersinia mollaretii ATCC 43969);gi|71065418|ref|YP_(—)264145.1|putative pyruvate decarboxylase(Psychrobacter arcticus 273-4); gi|16761331|ref|NP_(—)456948.1|putativedecarboxylase (Salmonella enterica subsp. enterica serovar Typhi str.CT18); gi|93005792|ref|YP_(—)580229.1|Pyruvate decarboxylase(Psychrobacter cryohalolentis K5);gi|23129016|ref|ZP_(—)00110850.1|COG3961: Pyruvate decarboxylase andrelated thiamine pyrophosphate-requiring enzymes (Nostoc punctiforme PCC73102); gi|16417060|gb|AAL18557.1|AF354297_(—)1 pyruvate decarboxylase(Sarcina ventriculi); gi|15607993|ref|NP_(—)215368.1|PROBABLE PYRUVATEOR INDOLE-3-PYRUVATE DECARBOXYLASE PDC (Mycobacterium tuberculosisH37Rv); gi|41406881|ref|NP_(—)959717.1|Pdc (Mycobacterium avium subsp.paratuberculosis K-10); gi|91779968|ref|YP_(—)555176.1|putative pyruvatedecarboxylase (Burkholderia xenovorans LB400);gi|15828161|ref|NP_(—)302424.1|pyruvate (or indolepyruvate)decarboxylase (Mycobacterium leprae TN);gi|118616174|ref|YP_(—)904506.1|pyruvate or indole-3-pyruvatedecarboxylase Pdc (Mycobacterium ulcerans Agy99);gi|67989660|ref|NP_(—)001018185.1|hypothetical protein SPAC3H8.01(Schizosaccharomyces pombe 972h-);gi|21666011|gb|AAM73540.1|AF282847_(—)1 pyruvate decarboxylase PdcB(Rhizopus oryzae); gi|69291130|ref|ZP_(—)00619161.1|Pyruvatedecarboxylase: Pyruvate decarboxylase (Kineococcus radiotoleransSRS30216); gi|66363022|ref|XP_(—)628477.1|pyruvate decarboxylase(Cryptosporidium parvum Iowa II);gi|70981398|ref|XP_(—)731481.1|pyruvate decarboxylase (Aspergillusfumigatus Af293); gi|121704274|ref|XP_(—)001270401.1|pyruvatedecarboxylase, putative (Aspergillus clavatus NRRL 1);gi|119467089|ref|XP_(—)001257351.1|pyruvate decarboxylase, putative(Neosartorya fischeri NRRL 181); gi|26554143|ref|NP_(—)758077.1|pyruvatedecarboxylase (Mycoplasma penetrans HF-2);gi|21666009|gb|AAM73539.1|AF282846_(—)1 pyruvate decarboxylase PdcA(Rhizopus oryzae).

Alcohol dehydrogenases (adh) catalyze the final step of amino acidcatabolism, conversion of an aldehyde to a long chain or complexalcohol. Various adh genes are known in the art. As indicated hereinadh1 homologs and variants include, for example, adh2, adh3, adh4, adh5,adh 6 and sfal (see, e.g., SFA (Saccharomyces cerevisiae)gi|288591|emb|CAA48161.1|(288591); the sequence associated with theaccession number is incorporated herein by reference).

Citramalate synthase catalyzes the condensation of pyruvate and acetate.CimA encodes a citramalate synthase. Homologs and variants are known andinclude, for example, citramalate synthase (Leptospira biflexa serovarPatoc) gi|116664687|gb|ABK13757.1|(116664687); citramalate synthase(Leptospira biflexa serovar Monteralerio)gi|116664685|gb|ABK13756.1|(116664685); citramalate synthase (Leptospirainterrogans serovar Hebdomadis) gi|116664683|gb|ABK13755.1|(116664683);citramalate synthase (Leptospira interrogans serovar Pomona)gi|116664681|gb|ABK13754.1|(116664681); citramalate synthase (Leptospirainterrogans serovar Australis) gi|116664679|gb|ABK13753.1|(116664679);citramalate synthase (Leptospira interrogans serovar Autumnalis)gi|116664677|gb|ABK13752.1|(116664677); citramalate synthase (Leptospirainterrogans serovar Pyrogenes) gi|116664675|gb|ABK13751.1|(116664675);citramalate synthase (Leptospira interrogans serovar Canicola)gi|116664673|gb|ABK13750.1|(116664673); citramalate synthase (Leptospirainterrogans serovar Lai) gi|116664671|gb|ABK13749.1|(116664671); CimA(Leptospira meyeri serovar Semaranga)gi|119720987|gb|ABL98031.1|(119720987); (R)-citramalate synthasegi|2492795|sp|Q58787.1|CIMA_METJA(2492795); (R)-citramalate synthasegi|22095547|sp|P58966.1|CIMA_METMA(22095547); (R)-citramalate synthasegi|22001554|sp|Q8TJJ1.1|CIMA_METAC(22001554); (R)-citramalate synthasegi|22001553|sp1026819.1|CIMA_METTH(22001553); (R)-citramalate synthasegi|22001555|sp|Q8TYB1.1|CIMA_METKA(22001555); (R)-citramalate synthase(Methanococcus maripaludis S2)gi|45358581|ref|NP_(—)988138.1|(45358581); (R)-citramalate synthase(Methanococcus maripaludis S2) gi|44921339|emb|CAF30574.1|(44921339);and similar to (R)-citramalate synthase (Candidatus Kueneniastuttgartiensis) gi|91203541|emb|CAJ71194.1|(91203541), each sequenceassociated with the foregoing accession numbers is incorporated hereinby reference.

EXAMPLES

A cyanobacterium, S. elongates, was engineered as follows. The ketoaciddecarboxylase gene kivd from Lactococcus lactis was expressed using anexpression cassette under the control of theisopropyl-β-D-thiogalactoside (IPTG) inducible promoter Ptrc (FIG. 1C).This DNA fragment was integrated into neutral site I (NSI)14 byhomologous recombination, resulting in SA578 (FIG. 1D). To increase theflux to the keto acid precursor, 2-ketoisovalerate (KIV), the alsS genefrom Bacillus subtilis and the ilvC and ilvD genes from Escherichia coliwere inserted into neutral site II (NSII) of the SA578 genome, resultingin SA590. (FIG. 1E). All three enzyme assays of SA590 lysatesdemonstrated higher activity than those from SA578 (FIG. 1F), indicatingthat alsS (B. subtilis), ilvC (E. coli) and ilvD (E. coli) are expressedand functional in S. elongatus. Because the vapor pressure ofisobutyraldehyde is relatively high, it can be removed readily from theculture medium during production by the bubbling of air. Evaporatedisobutyraldehyde was then condensed with a Graham condenser.

The strain was cultured in a Roux culture bottle at 30° C.Isobutyraldehyde concentrations in the culture medium and the trap weremeasured. The trap was refreshed daily. The strain produced 723 mg/lisobutyraldehyde in 12 d with an average production rate of 2,500 μgl⁻¹h⁻¹ (FIG. 1G-H). This number is very encouraging, as it is alreadyclose to the benchmark. The isobutyraldehyde production rate remainedconstant for the first 9 d, but the production rate decreased after thetenth day (FIG. 1H-I). When the culture was resuspended in fresh mediumafter 10 d, the bacteria regained their productivity (˜60 mg l⁻¹ d⁻¹),suggesting that some inhibitory metabolites accumulated during thecultivation. As expected, during the production process theisobutyraldehyde concentration in the culture medium remained low,around 20 mg/l (FIG. 1I). This low concentration would reduce toxicityto cells and prolong the production phase. This strain did not produceisobutanol, indicating that endogenous alcohol dehydrogenase (ADH)activity toward isobutyraldehyde was not detectable.

Isobutyraldehyde can also be converted to isobutanol by cyanobacteria.Increasing attention has been paid to isobutanol as a potentialsubstitute for gasoline or as a chemical feedstock. Thus, it would beworthwhile demonstrating the biological feasibility of isobutanolproduction by cyanobacteria. To demonstrate the direct synthesis ofisobutanol, three alcohol dehydrogenases (ADH2 from Saccharomycescerevisiae, YqhD from E. coli, and AdhA from L. lactis) along with Kivdfrom L. lactis were used. Their corresponding genes were integrateddownstream of kivd (FIG. 1D) individually, resulting in strains SA413,SA561 and SA562. After KIV was added to the growth medium the reactionproducts isobutyraldehyde and isobutanol were detected (FIG. 1K). Amongthe three dehydrogenases tested, YqhD was the most active in S.elongatus (FIG. 1K). YqhD is an NADPH-dependent enzyme, whereas AdhA andADH2 are NADH-dependent. These results suggest that the NADH generatedin the cell was insufficient for the NADH-dependent ADH. To increase theflux to KIV, the amplified KIV pathway (FIG. 1E) was combined with thealcohol-producing pathway (Kind and YqhD). The strain (SA579) produced450 mg/l of isobutanol in 6 d (FIG. 1M-O and FIG. 1L).

The tolerance of S. elongatus to isobutyraldehyde and isobutanol wasalso measured (FIG. 1P). Wild-type S. elongatus was able to tolerateconcentrations of isobutyraldehyde up to 750 mg/l (FIG. 1Q), but showedgrowth retardation in the presence of the same concentration ofisobutanol. This result shows that isobutyraldehyde is less toxic to thecell than isobutanol. In addition, the isobutyraldehyde tolerance levelof S. elongatus is much higher than the concentration found in theculture medium during production. These data are consistent with theresult that the isobutyraldehyde production strain produced constantlyfor 9 d in this system. Thus, the in situ product removal systemeffectively avoids toxicity effects.

Although productivity (total product divided by volume and time) is notthe only factor that determines the potential of a production system,the productivities of the engineered cyanobacteria for isobutyraldehydeand isobutanol demonstrated here are already higher than theproductivites of cyanobacteria demonstrated for hydrogen or ethanol(FIG. 10). As producing biodiesel from microalgae has been proposed asone of the most efficient methods, the algal diesel productivity (1×10⁵liter ha⁻¹ per year, which corresponds to about 4,000 μl l⁻¹ h⁻¹assuming 1 m characteristic dimension) was used as a benchmark forisobutyraldehyde production. Although the productivity of lab-scaleexperiments cannot be directly translated to industrial-scaleproduction, our productivity of isobutyraldehyde (6,230 μg 1⁻¹ h⁻¹) isencouraging (FIG. 10). This result demonstrates the technicalfeasibility for direct conversion of CO₂ to fuels or chemicals, whichcould become an economically feasible option after further improvement.The strategy further expands the utility of photosynthesis and bypassesthe need for biomass deconstruction and may therefore provide analternative path for addressing two of humanity's most pressingproblems: energy and climate change.

Reagents. Restriction enzymes and Antarctic phosphatase were from NewEngland Biolabs. Rapid DNA ligation kit was from Roche. KOD DNApolymerase was from EMD Chemicals. Oligonucleotides were from EurofinsMWG Operon. The chemicals, ribulose-1,5-bisphosphate,ribulose-1,5-bisphosphate carboxylase, NADPH,2,4-dinitrophenylhydrazine, propionic acid, acetoin, 2-keto-isovalerateand cocarboxylase were obtained from Sigma-Aldrich. NaH₁₄CO₃ (specificactivity 5 mCi/mmol) was purchased from American Radiolabeled Chemicals.

Strains and plasmids construction. Strains and plasmids used in thiswork are described in the following table. The primers used are listedin the table below.

Table of strains and plasmids used in this study Strain Relevantgenotype Synechococcus strains PCC7942 wild-type SA413 kivd-ADH2integrated at NSI in PCC7942 chromosome SA561 kivd-yqhD integrated atNSI in PCC7942 chromosome SA562 kivd-adhA integrated at NSI in PCC7942chromosome SA578 kivd integrated at NSI in PCC7942 chromosome SA579alsS-ilvC-ilvD integrated at NSII in SA561 chromosome SA590alsS-ilvC-ilvD integrated at NSII in SA578 chromosome Plasmids pAM2991NSI targeting vector; Ptrc pMMB66EH IncQ; AmpR; Ptac pSA55 Co1E1 ori;AmpR; PL1acO1: kivd-ADH2 pSA65 Co1E1 ori; AmpR; PL1acO1: kivd-adhA pSA68Co1E1 ori; AmpR; PL1acO1: alsS-ilvC-ilvD pSA78 From pAM2991 withkivd-ADH2 pSA126 NSII targeting vector; carries PL1acO1::alsS-ilvC-ilvDpSA129 Co1E1 ori; AmpR; PL1acO1: kivd pSA138 Co1E1 ori; AmpR; PL1acO1:kivd-yqhD pSA149 From pAM2991 with kivd-adhA pSA150 From pAM2991 withkivd-yqhD pSA155 From pAM2991 with kivd

Table of synthetic oligonucleotides used in this study namesequence (SEQ ID NO:) A148 GCCACCGGTCTCCAATTCTATACAGTAGGAGATTACCTATTAG(1) A149 CGGGATCCTTATTTAGAAGTGTCAACAACGTAT(2) A217GGCGAGCTCCGATCGCTTTGGGACTTGGAACGGT(3) A218GGCGAGCTCAAATCACCAGCTGAAACGGTGAAGT(4) A219CGCCTAGGAACCGTTCCTGCGCGATCGCTCTTA(5) A220CGCCTAGGTAAGCGGGCCACGGCAGCGAAAGGG(6) A258CGGGATCCTTATTTAGTAAAATCAATGACCATT(7) A259CGGGATCCTTAGCGGGCGGCTTCGTATATACGG(8) A262CGGGATCCTTATGATTTATTTTGTTCAGCAAAT(9) A308GAGTGGCAATTGATGCCCAAGACGCAATCTGCCGCAG(10) A309GGTATATCTCCTTCTTTTAGAGCTTGTCCATCGTTTCGAAT (11) A310AACGATGGACAAGCTCTAAAAGAAGGAGATATACCATGAAAAC TCTGCCCAAAGAGCGTC(12) A311CGGGATCCTTAGTAGCGGCCGGGACGATGAACG(13) A315GGAAGATCTTTCGTGTCGCTCAAGGCGCACTCCC(14) A316GGAAGATCTGTCTTGCCACGCCGAGCACCTGGTC(15) A317CGGGATCCGATATCTGGCGAAAATGAGACGTTG(16) A318GGGCCTGCAGGATATCAAATTACGCCCCGCCCTGC(17)

The neutral site I (NSI) targeting vector. Strains that express kivd andadh were constructed by insertion of an expression cassette into NSI14.The genes kivd and adh were cloned into the NSI targeting vector,pAM2991, under the IPTG-inducible Ptrc promoter. The coding region ofkivd-ADH2, kivd-adhA, kivd-yqhD and kivd were amplified from pSA55,pSA65, pSA134 and pSA129, respectively, using oligonucleotides A148 andA149, A148-A258, A148-A259 and A148-A262, respectively. The resultingplasmids were named pSA78 (kivd-ADH2), pSA149 (kivd-adhA), and pSA150(kivd-yqhD) and pSA155 (kivd).

The neutral site II (NSII)17 targeting vector. Construction of pSA68,which contains alsS (B. subtilis)-ilvC-ilvD (E. coli), was constructedas previously described. To clone the 5′ fragment of NSII, genomic DNAof S. elongatus was used as the PCR template with primers A217 and A218.PCR products were digested with SacI and cloned into pSA68 cut with thesame enzyme, creating pSA117. A correct orientation of the fragment wasconfirmed by PCR. To clone the chloramphenicol resistance gene, pACYC184was used as the PCR template with primers A225 and A226. PCR productswere digested with SpeI and cloned into pSA117 cut with the same enzyme,creating pSA122. A correct orientation of the fragment was confirmed byPCR. To clone the 3′ fragment of NSII, genomic DNA of S. elongatus wasused as the PCR template with primers A219 and A220. PCR products weredigested with AvrII and cloned into pSA122 cut with the same enzyme,creating pSA126. A correct orientation of the fragment was confirmed byPCR.

Transformation of S. elongatus. Transformation of S. elongatus wascarried out as described. Cyanobacterial transformants with thetargeting vectors were selected on BG-11 agar plates supplemented withantibiotics as appropriate; 20 μg/ml spectinomycin, 10 μg/ml kanamycinand 5 μg/ml chloramphenicol. Results of the transformation wereconfirmed by PCR and enzyme assays.

Medium and culture conditions. Wild-type S. elongatus and mutant strainswere grown in a modified BG-11 medium with the following modifications:50 mM NaHCO₃ and 10 mg/l thiamine were added. For an experiment with 5%CO₂ bubbling, 50 mM NaHCO₃ was not added. Cyanobacterial cells weregrown at 30° C. under fluorescent light (55 μE s⁻¹m⁻²), which wasprovided by eight 86-cm 20-W fluorescent tubes placed 15 cm from thecell culture. Cell growth was monitored by measuring OD730 of eachculture.

Culture conditions for isobutanol and isobutyraldehyde production. Forisobutyraldehyde and isobutanol production, cells were grown in 600 mlmedium in 1,000-ml Roux culture bottles that were aerated by air or aircontaining 5% CO₂. The culture was allowed to grow at 30° C. to OD₇₃₀ of0.4-0.6, at which point 1 mM IPTG was added. Daily, one-tenth the totalvolume of cell culture was removed from the cell culture. Then the samevolume of fresh medium containing 0.5 M NaHCO₃ was added to cellculture. pH of cell culture with NaHCO₃ was adjusted to 7.5 with 10 NHCl everyday. Utilization of 5% CO₂ stabilized the pH of cell culturearound ˜7.0, thus the pH was not adjusted. Presumably, the constant pHis due to the balance between CO₂ dissolution and consumption.

Quantification of the products. The alcohol and aldehyde compoundsproduced were quantified by a gas chromatograph equipped with a flameionization detector. Other secreted metabolites were quantified by ahigh-performance liquid chromatography.

Preparation of Cell-Free Extracts. Cells were collected 24 h afterinduction by centrifugation (4,000 g, 10 min, 25° C.). For the Als, IlvCand IlvD assays, the cells were washed once in 1 mM MgCl₂ and 100 mM3-(N-morpholino) propanesulfonic acid (MOPS), pH 7.0, then resuspendedin the same buffer. The cells were broken by passage through a chilledFrench pressure cell at 20,000 p.s.i. (4° C.) for a total of threetimes. Total protein measurements were made with the Bradford proteinassay kit from Bio-Rad.

Als assay. The Als assay was performed as described previously, with theexception that the reaction mixture contained 20 mM sodium pyruvate, 100mM MOPS buffer, pH 7.0, 1 mM MgCl₂ and 100 μM cocarboxylase. Theconcentration of acetoin produced was determined by a standard curvecreated using pure acetoin. One specific unit of Als activitycorresponds to the formation of 1 nmol of acetoin per mg of solubleprotein per min at 37° C.

IlvC assay. To measure the reduction of 2-acetolactate to2,3-dihydroxy-isovalerate, the oxidation of NADPH was monitored by adecrease in absorbance at 340 nm. The substrate, 2-acetolactate, wasfirst produced in a separate reaction as described for the Als assayusing purified, heterogeneously expressed B. subtilis AlsS in E. colistrain BL21. From this reaction 180 μl was added to 200 mM potassiumphosphate buffer, pH 7.5, 4 mM MgCl2 and 0.1 mM NADPH for a finalreaction volume of 1 ml. The samples were incubated at 30° C. for 5 min,then the reaction was initiated with the addition of cell extracts.Absorbance was measured at 340 nm. IlvC activity is expressed as nmol ofNADPH oxidized per min per mg of soluble protein at 30° C.

IlvD assay. The IlvD assay was performed as described previously. The500 μl reaction mixture contained 5 mM MgSO₄, 50 mM Tris-C1, pH 8.0,cell-free extract and 10 mM 2,3-dihydroxy-isovalerate. The substrate,2,3-dihydroxy-isovalerate, was synthesized as described previously.After the reaction mixture was preincubated for 5 min at 37° C., thesubstrate was added to initiate the reaction. The samples were incubatedfor 15 min at 37° C. The reaction was terminated by the addition of 125μl of 10% trichloroacetic acid, then 250 μl of saturated2,4-dinitrophenylhydrazine in 2 N HCl was added to the samples. After 20min at 25° C., 875 μl of 2.5 N NaOH was added and then the samples wereincubated for another 30 min at 25° C. The samples were then spun downfor 1 min to remove coagulated protein. Sample absorbances were measuredat 550 nm. Standard curves were created from known amounts of KIV. Thespecific activity was calculated as 1 nmol of KIV synthesized per mg ofsoluble protein per min at 37° C.

O₂ production measurements. The S. elongatus cultures were similarlycultured and induced as they were for isobutyraldehyde production.Periodically, 2 ml culture samples were measured for OD₇₃₀ and O₂production using the Oxygraph System (Hansatech Instruments). Datapoints represent triplicate measurements.

Attached hereto and incorporated herein, in addition to the figures, aresequences that are relevant to the practice of the disclosure. Thesequences correspond to particular coding sequences and polypeptidesequence for enzymes useful in generating a biofuel. One of skill in theart can readily determine which sequence is appropriate for a referencedgene or homolog. For example, reference to kind, would include thesequence set forth in SEQ ID NO:18 (cDNA) and SEQ ID NO:19(polypeptide), variants comprising a percent identity and having adecarboxylase function and homologs from other organisms.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A method of producing isobutyraldehyde comprising: (a) providing arecombinant photoautotrophic microorganism in a culture medium, whereinthe recombinant microorganism expresses a metabolic pathway comprising:an acetolactate synthase, an acetohydroxy acid isomeroreductase, adihydroxyacid dehydratase, and a 2-ketoisovalerate decarboxylase,wherein one or more enzymes in the metabolic pathway is exogenous; (b)culturing the recombinant microorganism of (a) in the presence of CO₂,wherein isobutyraldehyde is produced via photosynthesis and themetabolic pathway of (a); and (c) removing the isobutyraldehyde from theculture medium.
 2. The method of claim 1, wherein the recombinantmicroorganism is a cyanobacterium.
 3. The method of claim 2, wherein thecyanobacterium is Synechococcus elongatus.
 4. The method of claim 1,wherein the acetolactate synthase is exogenous and obtained fromBacillus subtilis.
 5. The method of claim 1, wherein the acetohydroxyacid isomeroreductase is exogenous and obtained from Escherichia coli.6. The method of claim 1, wherein the dihydroxy dehydratase is exogenousand obtained from Escherichia coli.
 7. The method of claim 1, whereinthe 2-ketoisovalerate decarboxylase is exogenous and obtained fromLactococcus lactis.
 8. The method of claim 1, wherein theisobutyraldehyde is removed from the culture medium by gas bubbling. 9.The method of claim 1, wherein the isobutyraldehyde is removed from theculture medium by evaporation.
 10. The method of claim 9, furthercomprising condensing the evaporated isobutyraldehyde.
 11. The method ofclaim 1, wherein isobutanol is not produced by the recombinantmicroorganism at a detectable level.
 12. A recombinant photoautotrophicmicroorganism that produces isobutyraldehyde, wherein the recombinantmicroorganism expresses a metabolic pathway comprising an acetolactatesynthase, an acetohydroxy acid isomeroreductase, a dihydroxyaciddehydratase, and a 2-ketoisovalerate decarboxylase, wherein one or moreenzymes in the metabolic pathway is exogenous, and wherein therecombinant microorganism cannot produce detectable levels ofisobutanol.
 13. The recombinant microorganism of claim 12, wherein therecombinant microorganism is a cyanobacterium.
 14. The recombinantmicroorganism of claim 13, wherein the cyanobacterium is S. elongatus.15. The recombinant microorganism of claim 12, wherein the acetolactatesynthase is exogenous and obtained from B. subtilis.
 16. The recombinantmicroorganism of claim 12, wherein the acetohydroxy acidisomeroreductase is exogenous and obtained from E. coli.
 17. Therecombinant microorganism of claim 12, wherein the dihydroxyaciddehydratase is exogenous and obtained from E. coli.
 18. The recombinantmicroorganism of claim 12, wherein the 2-ketoisovalerate decarboxylaseis exogenous and obtained from L. lactis.
 19. A method of preparing arecombinant microorganism that produces isobutyraldehyde comprising:transforming a photoautotrophic microorganism with one or more targetingvectors comprising one or more polynucleotides encoding (i) one or moreof an acetolactate synthase, an acetohydroxy acid isomeroreductase, adihydroxyacid dehydratase, and a 2-ketoisovalerate decarboxylase; and(ii) at least one inducible promoter, to prepare a recombinantphotoautotrophic microorganism, wherein the recombinant photoautotrophicmicroorganism produces isobutyraldehyde and cannot produce isobutanol.20. The method of claim 19, wherein the photoautotrophic microorganismis a cyanobacterium.